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Laboratory  Guide 


IX 


Experimental  Pharmacology 


BY 


CHARLES  W.  EDMUNDS,  A.B.,  M.D. 

PROFESSOR    OF    MATERIA    MEDICA    AND   THERAPEUTICS 
IN  THE  UNIVERSITY  OF  MICHIGAN 

AND 

ARTHUR  R.  CUSHNY,  A.M.,  M.D.,  LL.D.,  F.R.S. 

PROFESSOR  OP   PHARMACOLOGY  IN    UNIVERSITY   COLLEGE, 

LONDON  ;    AND    LATE    PROFESSOR    OF    MATERIA, 

MEDICA    AND    THERAPEUTICS    IN    THE 

UNIVERSITY   OF    MICHIGAN 


George  Wahr 

publisher  and  bookseller 

ann  arbor,  mich. 


CoPYRIGHTj   1918 

GEORGE  WAHR 
Ann  Arbor,  Mich. 


THE   ANN  ARBOR  PRESS 
ANN  ARBOR 


PREFACE  TO  1918  EDITION. 

In  the  years  which  have  elapsed  since  these  notes  were 
first  put  into  permanent  form  the  book  has  been  placed  in 
service  in  different  pharmacological  laboratories  over  the 
country,  and  in  this  revision  which  the  progress  of  the  sci- 
ence makes  necessary,  the  suggestions  which  have  been  re- 
ceived from  teachers  of  the  subject  have  proved  most 
welcome  and  helpful.  It  would  seem  that  the  wish  ex- 
piressed  in  the  preface  to  the  first  edition  that  these  notes 
might  prove  useful  to  others  has  been  realized. 

It  need  hardly  be  pointed  out  that  the  object  of  the  book 
is  not  to  furnish  an  exhaustive  treatise  on  laboratory  meth- 
ods, but  rather  a  simple  outline  of  experiments  which  can 
easily  be  carried  out  by  the  average  medical  student  in  any 
laboratory  equipped  for  the  purpose.  That  the  arrange- 
ment is  entirely  practical  and  workable  is  proved  by  the  fact 
that  these  directions  essentially  as  here  published,  have 
been  in  use  in  this  laboratory  for  considerably  over  twenty 
years. 

The  more  important  changes  which  have  been  made  in 
this  revision  may  be  mentioned  shortly.  The  section  on 
pharmaceutical  work  has  been  enlarged  by  the  introduction 
of  directions  for  making  a  few  preparations  other  than  those 
already  included  in  the  earlier  edition  of  the  book,  and  all 
of  these  directions  have  been  collected  and  placed  in  the 
early  part  of  the  book.  This  will  allow  of  the  omission  of 
this  section  entirely  in  schools  in  which  medical  students  are 
given  a  special  course  in  pharmacy.  In  case  no  separata 
course  in  this  subject  is  given  the  students,  the  devotion  of 


4  LABORATORY  GUIDE  IN 

a  few  hours  to  such  work  as  is  given  herein  wih  prove  very 
instructive. 

In  the  pharmacological  work  proper  a  section  on  biological 
assays  has  been' introduced,  and  in  addition  directions  for 
work  on  isolated  tissues  and  organs.  Attention  might  be 
called  also  to  the  experiment  on  the  action  of  "Digitalis  in 
auricular  fibrillation"  on  account  of  its  importance  from  the 
clinical  standpoint.  The  number  of  drugs  studied  has  not 
been  increased  as  it  has  been  felt  that  if  the  student  worked 
out  thoroughly  the  action  of  one  drug  as  typical  of  a  group 
he  could  work  out  the  other  members.  Such  an  arrange- 
ment tended  to  simplicity  and  to  economy  in  time  and  ani- 
mals. 

Finally,  attention  may  be  called  to  the  fact  that  in  the 
earlier  pharmacological  work  the  use  of  apparatus  is  re- 
duced to  a  minimum,  the  effect  of  this  being  to  train  the 
student's  powers  of  observation.  The  making  of  accurate 
protocols  of  the  experiments  in  the  laboratory  should  be 
emphasized  as  much  as  the  making  of  good  clinical  notes 
in  the  hospital,  and  the  former  may  v;ell  serve  as  a  train- 
ing for  the  latter. 

C.  W.  Edmunds. 
Ann  Arbor,  Mich., 

Jan.  1st,  1918. 


PREFACE  TO  THE  FIRST  EDITION 

The  following  course  has  been  gradually  developed  in  the 
pharmacological  laboratory  of  the  University  of  Michigan, 
and  it  has  been  suggested  that  if  put  in  a  more  permanent 
form  than  hitherto  it  might  be  helpful  to  other  teachers  of 
this  subject. 

The  practical  course  has  hitherto  been  given  as  an  intro- 
duction to  the  didactic  lectures  and  it  has  been  found  advis- 
able to  limit  the  number  of  drugs  examined  to  those  possess- 
ing the  most  typical  action.  The  object  has  been  to  train  the 
student  entering  on  the  subject  in  the  method  of  work  and  to 
impress  on  him  that  the  study  of  drugs  is  to  be  approached 
in  the  same  objective  way  as  other  branches  of  medicine. 

The  order  in  which  the  experiments  are  arranged  is  a 
purely  arbitrary  one,  necessitated  by  the  conditions  of  the 
laboratory,  and  can  doubtless  be  modified  with  advantage  to 
suit  other  classes. 

A  few  days  are  devoted  to  purely  pharmaceutical  work, 
but  the  chief  weight  is  laid  on  the  experimental  part  of  the 
course.  The  few  hours  devoted  to  pharmacy,  however,  train 
the  student  in  observing  the  characters  of  the  much  larger 
number  of  drugs  met  in  the  lecture  course,  and  have  been 
found  to  be  of  considerable  value.  A  beginning  may  be 
made  in  prescribing  as  occasion  offers,  but  this,  as  well  as  the 
therapeutic  applications  of  the  results  obtained  in  the  phar- 
macological experiments,  is  better  left  to  the  teacher  than 
expounded  in  the  laboratory  directions. 

Arthur  R.   Cushny. 
CHARiviiS   W.    Edmunds. 

Ann  Arbor,  March  i8th,  1905. 


!!('  !  \' 


CONTENTS. 

The  Pharmacopoeia   9 

General  directions  for  anaesthetics  and  operations.  ...  ii 

Physiological  solutions  for  animal  tissues 19 

Chemistry  of  drugs '. 20 

Percolation    44 

Powders    ' 5  ^ 

Capsules    55 

Cachets   59 

Pills 60 

Emulsions    67 

Ointments    72 

Suppositories    . 76 

Physiology  of  frog's  nervous  system 80 

Soporifics  or  Hypnotics 88 

Nux  vomica 96 

Camphor  group  112 

Opium    116 

Curara   127 

Nicotine 132 

Veratrine    ■ 135 

Caffeine 136 

Cocaine    143 

Belladonna   147 

Cinchona 156 

Anaesthetics    . 160 

Digitalis  series 168 

Aconite   183 

Drugs  on  the  frog's  heart 187 

Drugs  on  the  turtle's  heart 191 


8  CONTEXTS. 

Drugs  on  the  blood  pressure 200 

Drugs  on  the  mammahan  heart 207 

Analysis  of  tracings 215 

Digitalis  in  Auricular  Fibrillation 220 

Perfusion  of  blood  vessels   227 

Action  of  drugs  on  isolated  tissues 232 

Diuresis   239 

Drugs  on  the   sympathetic  system  and  on   intestinal 

peristalsis    247 

Salivary  and  pancreatic  secretion 255 

Antipyretics 267- 

Biological   assay    272 

Pilocarpus 283 

Nitrites 287 

Suprarenal  extract   288 

Pituitary  extract 291 

Intestinal  peristalsis   292 

Vasomotor  effects  in  the  intestine 299 

Vasomotor  effects  in  the  kidney 304 

Perfusion  of  isolated  frog's  heart 311 

Perfusion  of  isolated  mammalian  heart 315 

Drugs  and  reagents  required 317 


The  Pharmacopoeia. 

When  a  physician  prescribes  a  certain  drug  from  time  to 
time  in  his  practice  it  is  of  greatest  importance  to  him  to 
know  that  his  patients  are  always  getting  the  same  prepara- 
tion, containing  the  same  active  principles  in  uniform 
strength.  These  requirements  could  not  be  carried  out  if 
there  were  not  some  standard  recognized  by  the  manufac- 
turers and  followed  by  them  in  the  preparation  of  the  vari- 
ous medicinal  remedies.  Most  countries  possess  such  a 
standard  in  their  "Pharmacopoeias,"  which  are  generally 
published  by  the  government.  In  this  country,  the  govern- 
ment does  not  issue  the  pharmacopoeia,  but  it  recognizes  its 
authority.  The  book  is  revised  every  ten  years  by  a  Commit- 
tee of  Revision  composed  of  members  appointed  or  elected 
by  a  convention  of  various  medical  and  pharmaceutical  so- 
cieties and  colleges. 

It  need  hardly  be  remarked  that  in  such  a  work  only  such 
remedies  are  included  as  have  become  well  established. 
These  are  designated  as  "ofhciaF'  remedies,  while  drugs  not 
mentioned  are  "non-ofhcial." 

The  pharmacopoeia  gives,  first,  the  Latin  title  of  the  drug- 
followed  by  the  English  name,  and  the  official  abbreviation, 
and  in  the  case  of  chemicals  the  formula  and  molecular 
weight  are  given.  Any  synonyms  are  mentioned  and 
wherever  it  is  necessary,  a  short,  concise  definition  of  the 
drug  is  added,  telling  its  nature  and  its  source.  The  book 
then  gives  tests  by  which  the  identity  of  the  drug  may  be 
recognized,  and  its  purity  and  strength  tested  and  finally 
mentions  in  what  ways  and  in  what  doses  it  may  be  ad- 
ministered. 


lo  LABORATORY  GUIDE  IN 

Official  preparations  are  therefore  made  according  to  the 
directions  given  in  the  pharmacopoeias  and  they  should 
possess  uniform  strength  and  properties ;  such  an  ideal, 
however,  is  hard  to  attain,  as  some  of  the  crude  drugs,  even 
when  collected  according  to  pharmacopoeia!  directions,  dif- 
fer in  their  content  of  the  active  constituents. 


.  BXPBRIMBNTAL  PHARMACOLOGY  n 

General  Directions. 

Before  giving  the  specific  directions  for  the  experimental 
work  a  number  of  general  directions  are  given  for  the  com- 
moner operations  which  are  carried  out  on  animals  some  of 
which  are  usually  necessary  in  subsequent  manipulations. 

The  various  anaesthetics  employed  in  experimental  work 
are  also  described. 

Anaesthetics. 

No  animal  is  to  be  operated  on  unless  it  is  under  complete 
anaesthesia.  Ether  and  chloroform  may  be  used  for  this  pur- 
pose, but  have  the  disadvantage  of  requiring  constant  atten- 
tion. They  are  therefore  only  used  to  supplement  other 
anaesthetics  which  experience  has  shown  can  be  used  to  give 
full  anaesthesia  in  animals  without  the  necessity  of  their  be- 
ing continually  administered.  For  this  purpose  the  follow- 
ing combinations  of  drugs  usually  give  very  good  results,  al- 
though in  rare  cases  chloroform  or  ether  may  have  to  be 
given  in  addition. 

Anccsthctic  for  Rabbits. 

Paraldehyde,  1.7-2.00  cc.  per  Kg.  of  body  weight. 

While  your  assistant  holds  up  the  animal  by  all  four  legs 
and  head,  place  a  gag  in  the  mouth  and  pass  a  stomach  tube^ 
through  the  opening  in  the  gag,  being  very  careful  not  to 
pass  it  into  the  lungs.  Draw  the  paraldehyde  into  a  pipette 
and  place  the  point  of  the  pipette  in  the  opening  of  the 
stomach  tube  and  blow  the  drug  gently  into  the  stomach. 


*  A  soft  rubber  catheter  serves  very  well  as  a  stomach  tube  for  rabbits  and 
cats,  a  size  of  lo  or  15  F.  beinR  most  satisfactory.  These  catheters  will  pass 
mcire    easily   if   they    are   first    moistened   with    water. 


12 


LABORATORY  GUIDE  IN 


and  then  withdraw  the  tube.  If  after  waiting  15  minutes 
the  animal  is  not  completely  anaesthetized  ether  may  be  ad- 
ministered cautiously  or  a  second  small  injection  of  par- 
aldehyde given. 

In  place  of  paraldehyde  urethane  in  doses  of  i  G.  per 
Kg.  of  body  weight  may  be  used  as  an  anaesthetic.  The 
drug  is  dissolved  in  water  and  given  by  the  stomach  tube. 

Anesthetic  for  Cats. 

Chloretone,  0.3  G.  -  0.4  G.  per  Kg.  of  body  weight. 

Dissolve  the  chloretone^  in  as  small  a  volume  of  alcohol 
as  possible  and  add  about  an  equal  volume  of  water.  Place 
the  animal  in  a  cat  box-  and  administer  the  chloretone  so- 
lution by  means  of  the  stomach  tube.  The  stomach  tube 
is  passed  through  a  hole  in  the  center  of  a  gag  which  is 
firmly  held  between  the  jaws  of  the  animal  by  an  assistant. 

Anccsfhetic  for  a  Dog. 

Morphine  sulphate,  o.i  G.  to  0.25  G.  Chloretone,  3  G. 
to  4  G. 

•  One  or  two  hours  before  the  animal  is  needed  inject  the 
morphine  solution  under  the  skin  by  means  of  a  hypodermic 
syringe. 


^  It  is  convenient  to  keep  a  stock  solution  of  chloretone  in  95%  alcohol  on 
hand  so  that  the  required  amount  of  drug  may  be  measured  out  quickly.  A 
useful  strength  is  the  one  in  which  one  gram  of  chloretone  is  contained  in 
each  3   cc.   of  the  finished   solution. 

^  A  cat  box  is  of  the  greatest  service  as  a  means  of  protection  to  the  op- 
erator whenever  this  animal  is  used.  It  consists  of  an  ordinary  wooden  box 
about  30  or  35  cm.  long,  18  cm.  wide  and  15  or  18  cm.  deep.  It  is  provided 
with  a  sliding  cover,  which  has  a  V  shaped  cut  in  the  middle  of  one  end.  In 
the  corresponding  end  of  the  box  a  second  V  cut  is  made.  When  the  lid 
is  shut  these  two  cuts  leave  enough  room  for  the  animal's  neck.  The  lid  is 
kept  closed  tightly  by  means  of  a  nail  passed  through  the  opposite  end  of  the 
cover  into   the  end  of  the  box. 


.  EXPERIMENTAL  PHARMACOLOGY 


13 


Just  before  the  operation,  dissolve  the  chloretone  in 
alcohol  and  add  about  an  equal  amount  of  water  and  give 
it  to  the  dog  by  means  of  a  stomach  tube^  passed  through 
a  wooden  gag.  Another  very  satisfactory  method  of  giv- 
ing the  chloretone  is  to  dissolve  the  drug  in  5  or  10  cc.  of 
olive  oil  which  may  be  warmed  to  hasten  solution.  When 
dissolved  it  can  be  drawn  up  into  a  large  veterinary  all- 
metal  syringe.  While  the  animal  is  lying  on  its  side  the 
abdominal  wall  can  be  punctured  quickly  and  the  oily  solu- 
tion injected  into  the  peritoneal  cavity.  Anaesthesia  is  pro- 
duced in  about  10  minutes  by  this  method. 

Operations  on  Frogs. 

Pit]  ling. 

Before  exposing  the  heart  of  a  frog  or  carrying  out  any 
other  operation  upon  the  animal  its  brain  is  destroyed 
(pithed)  in  the  following  manner:  Hold  the  frog  in  the 
left  hand  with  its  back  upward,  tip  the  head  forward  with 
the  left  index  finger,  and  feel  for  the  prominent  angle  made 
at  the  junction  of  the  skull  and  spinal  column.  Just  back 
of  this  angle  make  a  cut  with  small  scissors  through  the 
skin  in  the  median  line.  Now  push  the  sharpened  end  of 
a  match  forward  through  the  foramen  magnum  into  the 
skull,  rotating  it  in  the  cranial  cavity  to  destroy  the  brain. 

Injecting  into  a  LympJi  Sac. 

In  the  intact  animal,  drugs  to  be  tested  are  usually  in- 
jected into  the  anterior  lymph  sac.  This  is  carried  out  in 
the  following  manner:  Lay  the  animal  back  downwards  in 


^  The   average   stomach  tube,   such  as   is  used  in  clinical   medicine,   is  about 
the   right  size   for  a  dog.     It  should  be  about   75   cm.   long. 


14  LABORATORY  GUIDE  IN 

the  palm  of  the  left  hand.  Hold  one  of  its  forelegs  firmly 
between  the  thumb  and  index  finger  and  the  other  foreleg 
between  the  middle  and  ring  fingers.  Draw  its  hind  legs 
downward  and  hold  them  against  the  palmar  surface  of 
the  hand  by  means  of  the  little  finger. 

Having  the  drug  in  the  glass  injecting  pipette/  which  is 
held  in  the  right  hand,  force  the  animal's  mouth  open  with 
the  point.  Pass  the  pipette  into  the  mouth  avoiding  ihe 
tongue,  which  is  attached  anteriorly  and  direct  the  point  to  • 
Vs'ard  the  floor  of  the  mouth  which  with  a  little  pressure  it 
will  pierce,  entering  the  lymph  sac.  As  it  is  pushed  down 
the  sac  the  point  can  be  seen  beneath  the  skin  of  the  ab- 
dominal wall.  The  finger  is  now  removed  from  the  upper 
end  of  the  pipette  and  the  drug  allowed  to  flow  into  the  sac, 
cr  if  necessary  blown  in. 

In  case  the  anterior  lymph  sac  has  been  destroyed,  for 
instance,  when  the  heart  has  been  exposed,  the  point  of  the 
pipette  may  be  pushed  onward  and  made  to  enter  either  one 
of  the  leg  lymph  sacs  or  one  of  the  lateral  sacs. 

To  Expose  the  Heart. 

Pith  the  frog  and  tie  it  on  its  back  on  the  frog  board. 
With  a  pair  of  forceps  raise  the  skin  in  the  median  line  of 
the  body  just  below  the  point  of  the  sternum  and  make  a 
nick  in  it  with  a  pair  of  scissors.     Starting  at  this  point 


iTo  make  a  glass  injection  pipette  take  a  piece  of  glass  tubing  about  7  mm. 
in  diameter  and  about  20  or  30  cm.  long  and  heat  the  middle  point  in  the  Bun- 
sen  flame,  rotating  the  tube  constantly,  and  when  it  is  red  hot  remove  it  from 
the  flame  and  draw  it  out  to  a  capillary  size;  cut  it  in  the  middle  so  as  to  leave 
a  fine  tube  about  8  cm.  long.  Heat  the  large  end  of  the  pipette  to  smooth  it 
oft  so  it  will   not  cut  the  tongue  or  lips. 


EXPERIMENTAL  PHARMACOLOGY 


15 


cut  up  and  outward  on  each  side  as  far  as  the  pectoral  girdle 
and  turn  this  V-shaped  piece  of  skin  upward  toward  the 
head.  Raise  the  sternum  by  placing  the  forceps  beneath 
the  point  and  with  scissors  cut  it  through  its  entire  length 


1 

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I. 

M. 

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p^w***^    '  ' 

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Fig.    Xo.    i. 


avoiding  injury  to  the  underlying  heart.  Tighten  the  strings 
on  the  fore  legs  so  as  to  pull  the  sides  of  the  sternum  well 
apart.  The  heart  is  now  seen  enclosed  in  the  pericardium, 
which  may  be  carefully  opened  and  cut  away. 


i6  LABORATORY  GUIDE  IN 

Directions  for  Operations  on  Mammals. 

The  following  are  the  most  common  operations  necessary 
in  experimental  pharmacology. 

To  Insert  a  Venous  Cannula. 

This  is  to  allow  of  the  intravenous  injection  of  drugs. 

Two  sizes  of  glass  venous  cannulas  (with  rubber  tubing 
attached)  are  shown  in  Fig.  No.  2.  The  larger  is  best  suited 
for  a  dog,  while  the  smaller  is  used  in  a  rabbit  or  cat.  Vari- 
ous sizes  should  be  provided. 


Fig.    No.   2. 

Operation.  When  the  anaesthesia  is  complete  make  an  in- 
cision through  the  skin  over  the  site  of  the  vessel  and  deepen 
the  cut  through  the  fascia  until  the  vein  is  exposed.  Avoid- 
ing injury  to  the  vessel  from  forceps  or  tearing,  clear  about 
two  inches  of  the  vein,  removing  all  the  fascia  surrounding 
it  so  as  to  leave  it  entirely  free.  Pass  two  threads  about  a 
foot  long  under  the  vein  and  place  "bull-dog"  forceps  on  it 
toward  the  cardiac  end  of  the  cleared  portion.  This  allows 
distention  of  the  vessel  by  the  blood.  Tie  one  of  the  threads 
tightly  around  the  upper  end  of  the  cleared  portion.     Hold 


EXPERIMENTAL  PHARMACOLOGY 


17 


the  end  of  this  Hgature  in  the  left  hand  and  place  the  left 
index  finger  under  the  vessel,  thus  making  it  tense.  With 
sharp  scissors  make  a  cut  in  the  distended  vein  and  place  the 
tip  of  the  cannula  in  it,  and  tie  it  in  place  with  the  loose 
thread  previously  placed  around  the  vessel.  Fill  the  cannula 
and  vein  with  salt  solution  (0.8  %),  taking  great  care  that 
,all  air  is  expelled. 

Precaution.  When  drugs  are  injected  intravenously  it  is 
of  the  utmost  importance  to  see  that  no  air  is  allowed  to 
f^nter  the  veins.  A  small  bubble  of  air  is  almost  invariably 
fatal  to  rabbits.  Be  sure  the  cannula  and  syringe  have  all 
the  air  expelled. 

To  Insert  an  Arterial  Cannula} 

This  is  to  allow  records  of  arterial  blood  pressure  to  be 
taken. 

Operation.  Expose  and  clear  the  artery,  usually  the 
■carotid,  in  the  manner  described  above  for  the  vein.  Pass 
two  ligatures  under  the  vessel  and  tie  the  thread  which  is 
■distal  to  the  heart.  Now  clamp  the  artery  with  the  "bull- 
dog" forceps,  placing  them  on  the  "heart  end"  of  the  cleared 
portion.  Place  the  index  finger  under  the  vessel  as  before 
and  make  a  cut  in  it  and  tie  a  cannula  in  place  with  the  loose 
ligature.  This  cannula  is  to  be  filled  with  sodium  sulphate 
solution  to  prevent  clotting  of  the  blood.- 


1  Arterial  cannulas  are  exactly  like  venous  cannulas.      Fig.   No.   2. 

2  Various  solutions  may  be  used  to  prevent  coagulation  of  the  blood.  Some 
•of  the   best  are   as  follows: 

Sodium  Sulphate,  half  saturated.  This  is  very  satisfactory  and  it  is  non- 
toxic, but  it  is  rather  dirty  in  case  some  is  spilled. 

Sodium  citrate,   5-10%. 

Magnesium  sulphate,  25%.  Very  satisfactory  but  must  be  used  with  care 
as  it  is  quite  toxic  in  case  some  is  allowed  to  enter  the  heart. 


i8  LABORATORY  GUIDE  IN 

To  Insert  a  Tracheal  Cannula. 

A  tracheal  cannula  is  inserted  to  allow  of  artificial  respira- 
tion being  carried  on  in  case  the  chest  is  to  be  opened  or  if 
the  anpesthesia  is  so  deep  that  the  respiratory  center  is 
seriously  depressed  or  paralyzed. 


Fig.  No.  3.     Glass  tracheal  cannula  with   rubber  tubing  attached. 

A  convenient  form  of  tracheal  tube  is  shown  in  Fig  3. 
They  can  be  made  of  various  sizes  of  glass  tubing  so  as  to 
fit  the  trachea  of  any  animal.  When  in  use  the  piece  of  rub- 
ber tubing  shown  in  the  cut  is  partially  constricted  to  give 
tlie  proper  degree  of  inflation  to  the  lungs. 

Operation.  Make  a  .median  incision  in  the  neck  through 
the  skin  and  fascia  beginning  just  below  the  thyroid  cartil- 
age. Expose  the  trachea  by  separating  the  muscles  with  the 
fingers  or  blunt  hooks  and  dissect  away  all  structures  from 
around  it.  Pass  a  strong  thread  under  it  and  then  make  a 
transverse  cut  with  the  scalpel  between  two  rings  about  two- 
thirds  of  the  way  through.  Into  this  opening  pass  the 
tracheal  tube  and  tie  it  in  place  with  the  loose  ligature. 


EXPERIMENTAL  PHARMACOLOGY  19 

Physiological  Solutions  for  Animal  Tissues. 

Physiological  Salt  Sokition  for  Frogs 0.65% 

For  Mammals    0.9  % 

Ringer's  Solution  for  Frogs : 

NaCl   0.6  % 

KCl   0.0075 

CaClo  (crystals)   0.026 

NaHCOg o.oi 

Locke's  Solution  for  Mammals : 

NaCl 0.92% 

KCl   0.42 

CaClo  (crystals)   0.024 

NaHCOg    0.015 

Dextrose    o.i 

Tyrode's  Solution: 

NaCl   0.8% 

KCl   0.02 

CaClo    (crystals)    0.02 

NaHCOg   0.1 

Na^HPO^    0.005 

MgClo   0.01 

Glucose    0.1 


20  LABORATORY  GUIDE  IN 

Chemistry  of  Drugs. 

Alkaloids. 

Among  the  most  important  of  the  organic  compounds  em- 
ployed in  medicine  is  the  group  of  vegetable  bases  known  as 
Alkaloids.  They  are  found  in  almost  all  parts  of  plants,  but 
in  greatest  abundance  in  the  seeds  and  roots.  They  consti- 
tute in  almost  all  cases  the  active  constituents  of  the  plants 
in  which  they  are  found  and  some  of  them  are  among  the 
most  powerful  poisons  known. 

The  alkaloids  contain  carbon,  hydrogen,  nitrogen,  and 
usually  oxygen,  but  in  a  few  instances  the  latter  is  not  pres- 
ent. 

In  their  chemical  properties  they  resemble  ammonia  very 
closely,  having  an  alkaline  reaction  and  uniting  with  acids 
without  the  elimination  of  hydrogen  to  form  salts.  These 
salts  differ  from  the  alkaloids  in  their  solubility,  and  upon 
this  fact  is  based  one  method  of  isolating  them  in  a  pure 
form  from  the  plants  in  which  they  are  found. 

One  of  the  most  familiar  alkaloids  is  quinine,  which  is 
derived  from  various  species  of  cinchona  bark,  and  it  may 
be  taken  as  a  type  of  the  group,  as  the  reactions  given  by  it 
are  characteristic  of  most  of  the  other  alkaloids. 

Taste  quinine  and  quinine  sulphate. 

I.  Test  the  solubility  of  a  few  crystals  of  quinine 
(alkaloid)  and  of  quinine  sulphate  (alkaloidal  salt)  in  lo 
cc.  of  each  of  the  following: 


BXPBRIMENTAL  PHARMACOLOGY  21 


22 


LABORATORY  GUIDE  IN 


EXPERIMENTAL  PHARMACOLOGY 


23 


Solvent. 


Alkaloid  solubility. 


Alkaloidal  salt  solubility. 


Water. 
Alcohol. 
Ether. 
Chloroform. 


II.  Add  about  100  cc.  of  water  to  0.2  G.  quinine.  Shake 
the  flask  and  observe  only  a  small  part  goes  into  solution. 
Test  the  reaction  of  the  solution  with  litmus.  How  do  you 
explain  the  reaction? 

Add  I  cc.  dilute  sulphuric  acid  and  shake  until  complete 
solution  takes  place.  The  sulphuric  acid  formed  the  bisul- 
phate  of  the  alkaloid.  (Note  the  fluorescence  of  the  solution 
which  is  characteristic  of  quinine.)  Use  this  solution  in 
making  the  following  tests. 

III.  To  5  cc.  of  the  solution  add  slowly  potassium  car- 
bonate solution  until  a  distinct  alkaline  reaction  is  obtained. 
What  happens?  Render  the  mixture  acid  with  dilute  sul- 
phuric acid. 

Alkaloidal  salts  are  very  feeble  bases  and  are  thrown  out 
of  solution  by  the  addition  of  the  fixed  alkalies  and  their 
carbonates  which  unite  with  the  acid  of  the  alkaloidal  salt. 
The  precipitate  therefore  was  the  freed  alkaloid  which  is 
insoluble  in  water  and  which  on  the  addition  of  the  acid 
formed  the  sulphate  (or  bisulphate)  again,  which  passed 
into  solution. 

IV.  To  5  cc.  of  the  solution  add  potassium  hydroxide 
until  the  reaction  is  alkaline.  Add  distilled  water  until  the 
test  tube  is  nearly  full  and  then  shake  the  mixture  and  see  if 
the  precipitate  dissolves. 


24 


LABORATORY  GUIDE  IN 


The  precipitate  here,  as  in  No.  HI,  is  the  free  alkaloid. 
Ihe  alkaloids  are  spoken  of  as  being  insoluble  in  water,  but 
this  is  only  relatively  correct,  as  they  are  soluble  in  a  very 
large  excess ;  in  the  case  of  quinine  in  t6oo  parts  of  cola 
water. 

In  the  first  parts  of  experiments  III  and  IV  it  was  seen 
that  an  alkaloidal  salt  is  thrown  out  of  solution  by  the  fixed 
alkalies  and  their  carbonates.  For  this  reason  they  should 
not  be  prescribed  together  in  the  same  solution,  because  the 
alkaloid  would  fall  to  the  bottom  of  the  bottle  and  even  if 
it  were  shaken  each  time  before  a  dose  was  taken  the  chances 
are  that  the  last  dose  in  the  bottle  would  contain  a  much 
larger  amount  of  alkaloid  than  was  intended,  and  alarming 
if  not  fatal  symptoms  might  result.  The  same  objection 
holds  in  regard  to  prescribing  alkaloids  and  tannic  acids  to- 
gether in  solution  (see  Exp.  V  a). 

This  incompatibility  of  alkaloids  and  alkalies  may  be  over- 
come by  the  presence  of  a  sufficient  amount  of  alcohol. 

V.  Put  5  cc.  of  the  solution  into  each  of  5  test  tubes  and 
add  slowly  small  quantities  of  the  following  reagents ;  note 
the  formation  of  any  precipitate  and  if  any  occurs  test  its 
solubility  by  adding  water  and  shaking  as  was  done  in  Exp. 
IV.    Tabulate  the  results. 


Reagent. 

Precipitate? 

Soluble 

in   water? 

a* 

Tannic    acid. 

b 

Picric  acid. 

c 

Iodine  in  potassium  iodide. 

d 

Mercury-potassium  iodide. 

e 

Phosphotungstic  acid. 

^  See  experiment  VI. 


MXPERIMBNTAL  PHARMACOLOGY  25 


2S  LABORATORY  GUIDE  IN 


EXPERIMENTAL  PHARMACOLOGY 


27 


The  precipitate  in  (a)  was  the  tannate  of  the  alkaloid;  in 
(b)  the  picrate ;  (c)  polyiodide  (usually);  (d)  double 
iodide  of  the  alkaloid  and  mercury;  (e)  phosphotungstate  of 
the  alkaloid.  These  five  reagents,  with  a  few  others,  of  which 
the  most  important  are  gold  chloride  and  platinum  chloride, 
are  known  as  the  "alkaloidal  precipitants"  and  they  are 
largely  used  for  the  detection  of  the  different  members  of 
this  group. 

VI.  Filter  the  precipitate  obtained  with  tannic  acid  in. 
Exp.  V  a,  and  wash  it  with  a  little  water  and  then  taste 
it  comparing  its  taste  with  that  of  quinine  sulphate.  Qui- 
iime  tannate  is  sometimes  known  as  tasteless  quinine. 

VII.  To  10  cc.  of  the  quinine  solution  add  potassium 
hydroxide  until  the  reaction  is  alkaline.  Now  add  5  to  10 
cc.  of  chloroform  and  placing  the  thumb  over  the  mouth  of 
the  test  tube  shake  it  vigorously  for  a  minute  or  two  or  until 
the  precipitate  disappears.    Set  it  aside  for  a  short  time  until 

,  the  chloroform  settles  in  the  bottom  of  the  tube  when  it  is  to^ 
be  drawn  off  from  the  water  by  means  of  a  glass  pipette  and 
put  into  a  clean,  dry,  evaporating  dish  and  set  aside  to  allow 
the  chloroform  to  evaporate.  Also  draw  off  some  of  the 
water  into  a  clean  test  tube  and  test  for  the  presence  of 
alkaloid  by  adding  a  few  drops  of  the  solution  of  tannic 
acid.     Explain  the  results. 

This  experiment  illustrates  one  of  the  methods  used  in 
separating  an  alkaloid  from  other  bodies  which  are  found 
present  with  it  in  the  crude  drug.  It  is  known  as  the  "Shak- 
ing out  process"  and  will  be  more  fully  described  later.  (See 
nux  vomica  and  belladonna.) 

VIII.  Boil  10  cc.  of  the  quinine  solution  for  a  few  min- 
utes with  a  cubic  centimeter  of  sulphuric  acid ;  neutralize 


28  LABORATORY  GUIDE  IN 

the  acid  with  potassium  hydrate  and  apply  Fehhng's  test  for 
glucose.^     Is  there  any  reduction? 

Absence  of  reduction  shows  no  glucoside  is  present.  Con- 
trast next  section  (Glucosides). 

Examine  specimens  of  other  alkaloids  and  their  salts : — 

Strychnine,  from  the  nux  vomica  bean  (Strychnos  nux 
vomica) . 

Morphine,  from  opium,  the  dried  juice  of  the  unripe  cap- 
sules of  the  poppy  (Papaver  somniferum). 

Caffeine,  from  the  berry  of  cofifee  (Coffea  Arabica),  and 
from  tea  leaves  (Thea  Chinensis). 

Atropine,  from  deadly  nightshade  (Atropa  Belladonna). 

Berberine,^  from  golden  seal  (Hydrastis  Canadensis). 

Coniine,-  from  the  fruit  of  the  poison  hemlock  (Conium 
maculatum) . 

Nicotine,-  from  tobacco    (Nicotiana  tabacum). 

Notice  that  the  names  of  alkaloids  end  in  "ine,"  in  L,atin 
*'ina,"  as  for  example,  strychnina,  morphina,  etc. 

Glucosides. 

Next  in  importance  to  the  alkaloids  among  the  vegetable 
poisons  rank  the  Glucosides.  These  are  substances  which 
break  up  when  acted  on  by  dilute  acids  or  ferments,  and 
yield  as  one  of  their  decomposition  products  a  sugar  which 
in  many  cases  is  glucose ;  in  fact,  glucosides  may  be  regarded 

^  Fehling's  test  for  glucose  is  carried  out  as  follows:  Mix  in  a  test  tube 
equal  quantities  (about  i  cc.)  of  two  solutions,  one,  a  solution  of  copper  sul- 
phate, and  the  other,  a  solution  of  potassium  hydroxide  and  Rochelle  salts. 
The  resulting  mixture  is  a  clear,  dark  blue  solution,  which  is  to  be  boiled 
and  the  solution  to  be  tested  added  to  it.  If  glucose  is  present  a  red  precipi- 
tate of  cuprous  oxide  will  be  thrown  down  after  the  solution  has  stood  lor  a 
short  time.  The  precipitate  may  be  clearly  seen  in  the  bottom  of  the  test  tube 
if   a   reducing  substance  is  present. 

''■  Berberine  is  one  of  the  few  colored  alkaloids. 

^  Coniine  and  nicotine  are  tlfe  two  most  important  liquid  alkaloids.  They  con- 
tain r.o  oxygen  and  are  volatile. 


■EXPERIMENTAL  PHARMACOLOGY 


29 


^o  LABORATORY  GUIDB  IN 


BXPBRIMBNTAL  PHARMACOLOGY 


31 


as  salts  of  sugar.  The  other  products  differ  in  the  different 
glucosides,  in  some  cases  being  a  volatile  oil,  in  others  an 
alkaloid,  etc. 

In  the  following  experiments  amygdalin  will  be  employed. 
It  is  a  glucoside  which  is  found  in  bitter  almonds,  laurel 
leaves  and  in  the  bark  of  the  Virginian  prune  or  cherry. 

Make  50  cc.  of  a  2%  solution  of  amygdalin  in  water. 

I.  Apply  Fehling's  test  for  glucose  (see  page  28,  foot- 
note) to  5  cc.  of  the  amygdalin  solution.  Is  a  reducing  sub- 
stance present? 

II.  Boil  10  cc.  of  the  amygdalin  solution  with  i  cc.  of 
sulphuric  acid  (dilute)  for  10  minutes.  Neutralize  with 
sodium  hydrate  and  apply  Fehling's  test.  Do  you  get  any 
reduction?     Compare  with  Exp.  I,  and  explain. 

III.  To  5  cc.  of  the  amygdalin  solution  add  some  saliva 
and  place  it  on  the  water  bath  at  40°  for  half  an  hour.  Test 
for  a  reducing  substance  as  in  i  and  2  and  explain  results. 

IV.  Put  about  5  cc.  of  the  amygdalin  solution  into  each 
of  5  test  tubes  and  test  each  with  the  alkaloidal  precipitants. 


Reagent. 


Tannic   acid. 

Picric    acid. 

Iodine    in   potassium   iodide. 

Mercury-potassium  iodide. 

Fhosphotvingstic    acid. 


Precipitate? 


V.  Pulverize  three  bitter  almonds  in  a  dry  mortar  and 
place  the  powder  in  a  small  beaker  and  add  about  10  cc. 
of  warm,  not  hot,  water  to  it.  Boil  three  more  bitter  almonds 
for  two  or  three  minutes  in  about  10  cc.  of  water  and  then 
pulverize  them  in  a  mortar  and  add  warm  water  as  in  the 


32 


LABORATORY  GUIDE  IN 


first  instance.  Set  both  mixtures  aside  for  a  short  time  and 
compare  the  odors  from  the  two  beakers. 

The  odor  found  is  due  to  the  oil  of  bitter  ahiionds  formed 
by  the  decomposition  of  the  amygdahn  through  the  action 
of  a  ferment,  emulsin,  also  present  in  the  almonds  and  act- 
ing in  the  presence  of  water.  This  oil  consists  of  benzalde- 
hyde  and  prussic  acid  and  is  therefore  extremely  poisonous. 

What  reaction  is  characteristic  of  the  glucosides? 

Examine  specimens  of  other  glucosides : — 

Salicin,  found  in  the  willow  and  poplar. 

Digitalin,  found  in  the  fox  glove  (Digitalis  purpurea). 

Digitoxin,  found  in  the  fox  glove  (Digitalis  purpurea). 

The  names  of  glucosides  end  in  "in,"  in  Latin  "inum,"  as 
for  example,  salicinum. 

Volatile  Oils. 

The  group  of  volatile  oils  contains  a  large  number  of 
preparations  which  are  extensively  employed  in  medicine. 
They  are  also  known  as  the  Bssential  or  Bthcrcal  oils.  They 
are  found  widely  distributed  in  nature  as  the  odor  of  the 
various  plants  and  flowers  is  due  to  them  or  to  their  oxidized 
products.  In  their  chemistry  they  differ  greatly  from  the 
group  of  fatty  or  fixed  oils  from  which  they  must  be  care- 
fully distinguished,  the  latter  being  non-volatile.  The  volatile 
oils  are  not  to  be  regarded  as  pure  substances,  like  the  alka- 
loids, but  rather  as  mixtures  of  varying  composition.  Most 
of  them  are  colorless  when  fresh  and  pure,  but  they  tend 
to  become  colored  after  long  exposure  to  light  and  air. 
In  odor  and  taste  they  differ  widely  from  one  another,  but 
in  both  cases  these  properties  are  generally  characteristic 
of  the  individual  oil.  The  majority  of  them  are  lighter  than 
water,  there  being  only  a  few  heavier. 


■BXPBRIMBNTAL  PHARMACOLOGY  33 


3^  LABORATORY  GUIDE  IN 


nXPERIMUNTAL  PHARMACOLOGY 


35 


As  a  typical  member  of  the  group  the  oil  of  turpentine  is 
to  be  employed  in  the  following  experiments.  As  a  type  of 
the  fatty  or  fixed  oils  cotton  seed  oil  will  be  used  to  illustrate 
some  of  the  differences  between  the  two  series. 

I.     Solubility  of  volatile  oils. 

Into  each  of  five  test  tubes  put  i  cc.  of  the  oil  of  turpen- 
tine and  then  add  5  cc.  of  the  following  reagents.  Shake 
and  note  whether  the  oil  is  soluble. 


Reagent. 

Solubility? 

a 

Water. 

b 

Alcohol. 

c 

Ether. 

d 

Chloroform. 

e 

Cotton  see'J   oil. 

II.     Solubility  of  fixed  oils. 

Into  each  of  5  test  tubes  place  i  cc.  of  cotton  seed  oil 
and  then  add  5  cc.  of  the  following  reagents.  Shake  and 
note  the  degree  of  solubility  as  in  I. 


Reagent. 

Solubility? 

a 

Water. 

b 

Alcohol. 

c 

Ether. 

d 

Chloroform. 

e 

Oil  of  turpentine. 

What   differences    in    solubility    exist    between    the    two 


series : 


The  two  groups  of  oils  resemble  one  another  in  leaving  a 
greasy  stain  when  dropped  on  paper,  but  that  from  the  fixed 
oil  is  permanent. 


36  LABORATORY  GUIDE  IN 

III.  a.  Put  a  drop  of  oil  of  turpentine  on  glazed  paper. 
Note  the  stain.  Hold  the  paper  from  six  inches  to  a  foot 
above  the  flame  of  the  Bunsen  burner  for  a  few  minutes. 
Result  ? 

b.  Repeat,  using  cotton  seed  oil,  being  careful  not  tp  use 
enough  heat  to  char  the  paper.  How  does  it  differ  from  the 
previous  experiment? 

IV.  Decomposition  product, 

a.  Heat  5  drops  of  the  oil  of  turpentine  in  a  dry  test  tube. 
Note  the  odor. 

b.  Heat  strongly  5  drops  of  cotton  seed  oil  in  a  dry  test 
tube.     Note  the  odor. 

In  the  latter  case  acrolein  is  formed  from  the  partial  de- 
composition of  the  fat  and  is  recognized  by  its  irritating 
and  disagreeable  odor  resembling  burnt  fat. 

V.  Add  I  cc.  oil  of  turpentine  to  5  cc.  water  and  shake 
the  mixture  well  for  a  minute  or  two.  Allow  the  oil  and 
water  to  separate,  and  then  draw  off  the  latter  with  a  pipette 
into  a  clean  test  tube.  Note  its  odor  and  taste,  which  are  due 
to  the  turpentine,  of  which  the  water  has  taken  up  a  small 
amount,  forming  a  preparation  recognized  in  the  Pharma- 
copoeia as  an  "Aqua."  These  waters  flavored  by  the  various 
volatile  oils  are  largely  used  to  give  a  pleasant  taste  and 
smell  to  medicines. 

The  "Syrupi"  are  solutions  of  sugar  and  water  to  which 
is  often  added  a  small  quantity  of  a  volatile  oil  as  in  the 
aquae. 

Spiritiis,  spirits,  are  solutions  of  a  volatile  oil  in  alcohol 
\^Exp.  lb.);  they  are  largely  used  in  medicine  as  flavoring 
agents. 


liXPBRIMBNTAL  PHARMACOLOGY 


37 


38 


LABORATORY  GUIDE  IN 


■EXPERIMENTAL  PHARMACOLOGY  39 

Examine  specimens  of  the  different  volatile  oils  named 
below  and  compare  them  with  the  oil  of  turpentine  as  re- 
gards odor,  taste,  color,  etc. 

Oleum  Menthse  Piperitse  (oil  of  peppermint). 
"       Gaultherise   (wintergreen  oil). 
"       Lavandulae  Florum  (oil  of  lavender). 
"       Eucalypti  (eucalyptus  oil). 
"       Caryophylli  (oil  of  cloves).    ■ 

Resins. 

Among  the  less  important  chemical  compounds  used  in 
iJiedicine  are  the  Resins.  This  is  a  miscellaneous  class  char- 
acterized by  the  smooth,  shining  fracture. 

Using  powdered  resin  test  its  solubility  as  follows : — 


Reagent.  Solubility? 


a 

Water. 

b 

Alcohol.                      1 

c 

Ether. 

d 

Chloroform. 

e 

Oil  of  turpentine. 

Add  water  to  b  and  explain  result. 
Examine  the  Resin  of  jalap. 

Resin  of  copaiba. 
Olcoresins  are  solutions  of  resins  in  volatile  oils. 
Examine  Turpentine. 

Burgundy  pitch. 

Copaiba. 
Balsams  are  like  oleoresins,  but  contain  also  benzoic  and 
cinnamic  acids. 

Examine  Balsam  of  Peru. 

Balsam  of  Tolu. 


40  LABORATORY  GUIDE  IN 

Glim  resins  are  mixtures  of  gums  and  resins,  containing 
usually  in  addition  some  volatile  oil. 

Examine  Asafoetida. 

Gums  are  amorphous  transparent  substances  belonging  to 
the  group  of  carbohydrates.  Some  dissolve  in  water  v^hile 
others  only  .swell  to  a  jelly.     They  are  insoluble  in  alcohol. 

Examine  Acacia. 

Tragacanth, 

Saponins. 

In  a  large  number  of  plants  are  found  certain  substances 
of  a  glucosidal  nature  which  because  of  close  chemical  and 
pharmacological  relationships  have  been  included  under  one 
group  known  as  Saponins  or  in  the  case  of  the  more  toxic 
ones,  Sapotoxins.  Chemically  many  of  them  may  be  ar- 
ranged in  a  series  CnH^n-sOio  ^^'^  they  all  possess  the  char- 
acteristic glucosidal  reaction,  being  decomposed  by  acids 
and  ferments  with  the  formation  of  a  reducing  substance. 

They  possess  the  property  of  forming  a  soap-like  solu- 
tion with  a  very  persistent  foam  when  even  very  dilute 
v/atery  solutions  are  shaken.  It  is  on  account  of  this  prop- 
erty that  the  plants  from  which  some  of  them  are  derived 
have  obtained  their  popular  names  such  as  Soapwort 
(Saponaria  officinalis)  ;  Soapbark  (Quillaja  Saponaria), 
etc. 

They  also  aid  in  the  emulsification  of  fats  and  in  the  sus- 
pension of  fine  powders  and  act  powerfully  in  dissolving  red 
.blood  cells. 

Using  a  2%  solution  of  saponin  make  the  following 
tests : — 


UXPBRIMBNTAL  PHARMACOLOGY  41 


42  LABORATORY  GUIDE  IN 


EXPERIMENTAL  PHARMACOLOGY  43 

I.  To  I  cc.  in  a  lest  tube  add  5  or  10  cc.  of  water.  Shake 
well  and  then  set  aside  noting  the  persistence  of  the  foam. 

II.  To  2  cc.  castor  oil  add  i  cc.  of  the  saponin  solution 
and  shake  well.  Now  add  about  an  equal  volume  of  water 
and  shake  thoroughly.  A  uniform  white  mixture  consisting 
of  the  free  fat  droplets  held  in  suspension  by  the  saponin 
results.  Set  the  emulsion  aside  for  a  short  time  and  see  if 
the  oil  separates.  Later  add  to  the  emulsion  about  half 
as  much  alcohol  and  after  shaking  set  the  mixture  aside  and 
see  if  any  change  results. 


44  LABORATORY  GUIDE  IN 

Percolation. 

The  U.  S.  Pharmacopoeia  gives  the  following  definition  of 
this  process :  "The  process  of  percolation  consists  in  sub- 
jecting a  comminuted  substance  or  mixture  of  substances, 
contained  in  a  vessel  called  a  percolator,  to  the  solvent  ac-. 
tion  of  successive  portions  of  a  liquid  termed  the  menstruum 
in  such  a  manner  that  the  liquid,  as  it  traverses  the  powder 
in  its  descent  to  the  receiver,  shall  extract  the  soluble  con- 
stituents and  pass  from  the  percolator  free  from  insoluble 
niatter." 

The  liquid  with  the  soluble  portions  of  the  drug  dissolved 
in  it  is  known  as  the  percolate. 

The  drug  to  be  percolated  is  usually  ground  to  a  fine 
powder  in  order  to  break  up  the  vegetable  cells,  etc.,  so  that 
the  menstruum  can  penetrate  it  easily,  and  the  menstruum  is 
allowed  to  pass  through  the  drug  only  very  slowly,  giving  it 
.time  to  dissolve  the  soluble  constituents.  To  still  further  aid 
this  penetration,  a  common  plan  is  to  moisten  the  powder 
with  a  small  quantity  of  the  menstruum  and  to  allow  it  to 
remain  tightly  covered  (macerate)  for  twenty-four  or  forty- 
eight  hours  before  beginning  the  percolation.  This  also 
gives  time  for  the  drug  to  swell,  as  is  likely  to  occur  in  some 
cases. 

Percolators^  are  usually  cylindrical,  or  conical,  and  vary 
in  size  with  the  amount  of  drug  to  be  exhausted.  The 
lower  orifice  is  to  be  closed  with  a  tightly  fitting  cork,  which 
is  best  put  in   from  the  inside,  but  this  is  not  necessary 


^  For  all  purposes  in  this  course  the  cylindrical  percolator  of  the  following 
dimensions  is  large  enough:  Length,  28.2  cm.;  internal  diameter  at  top,  3.7 
cm.;  capacity,  275  cc.  It  should  be  provided  with  an  ordinary  iron  stand  and 
ring,  the  latter  being  padded  with  cotton. 


BXPBRIMBNTAL  PHARMACOLOGY  45 


^6  LABORATORY  GUIDE  IN 


EXPERIMENTAL  PHARMACOLOGY  47 

with  the  small  percolators.  Through  this  cork  passes  a  small 
glass  tube  (5  mm.  in  diameter),  the  inner  end  of  the  tube 
being  flush  with  the  top  of  the  cork.  To  the  outer  end  of  the 
tube  is  fitted  a  piece  of  rubber  tubing.  One  method  of  regu- 
lating the  flow  of  the  percolate  is  to  have  this  tubing  of  suf- 
ficient length  to  reach  nearly  to  the  top  of  the  percolator  and 
to  have  the  free  end  fitted  with  another  glass  tube ;  the  latter 
can  be  raised  or  lowered  until  the  percolate  escapes  at  the 
desired  rate.  The  other  method,  which  probably  is  not  quite 
as  good,  is  to  have  the  rubber  tube  about  an  inch  long  and  to 
regulate  the  flow  by  means  of  a  small  screw  clamp. 

Having  fitted  the  cork  and  tubing  the  percolator  is  ready 
to  pack  if  the  drug  has  macerated  the  required  length  of 
time.  The  packing  is  done  by  means  of  a  plunger,  which  is 
made  by  taking  a  cork  with  a  diameter  about  half  as  great  as 
the  diameter  of  the  upper  opening  of  the  percolator  and 
sticking  into  it  a  glass  rod  which  is  a  little  longer  than  the 
depth  of  the  percolator. 

Place  in  the  bottom  of  the  percolator  a  small  piece  of  ab- 
sorbent cotton  and  press  it  loosely  in  place.  Only  a  small 
amount  of  cotton  should  be  employed,  as  its  only  use  is  to 
keep  the  powdered  drug  from  passing  down  and  clogging  up 
the  exit  tube.  Now  put  in  the  drug,  packing  it  uniformly  in 
place  with  the  plunger.  Do  not  use  much  force  or  the  drug 
will  be  so  tightly  packed  that  it  will  be  very  hard  for  the 
menstruum  to  pass  through.  Very  little  more  force  will  be 
required  than  would  be  given  by  dropping  the  plunger  from 
a  height  of  a  few  inches.  Experience  together  with  a  knowl- 
edge of  the  physical  and  chemical  constitution  of  the  differ- 
ent drugs  can  alone  teach  how  much  force  to  employ.  When 
the  drug  is  in  place  cover  it  with  a  piece  of  filter  paper  cut 


48  LABORATORY  GUIDE  IN 

so  as  to  roughly  fit  its  upper  surface  and  weight  it  down 
with  a  small  glass  stopper.  Pour  in  the  menstruum  to  the 
top  of  the  percolator  and  cover  the  latter  with  a  small  glass 
plate.  If  the  lower  orifice  is  open  the  menstruum  will  soon 
penetrate  through  the  drug  and  begin  to  drop  from  the  rub- 
ber tube.  In  cases  in  which  the  formula  prescribes  that  the' 
drug  is  to  macerate  for  a  certain  length  of  time  in  the  perco- 
lator, the  clamp  is  closed,  or  if  a  long  tube  has  been  used  the 
end  is  raised  to  a  level  which  will  prevent  any  outflow.  At 
the  end  of  the  prescribed  time  allow  the  percolate  to  escape, 
receiving  it  in  a  bottle  or  flask.  Regulate  the  outflow  so  that 
it  shall  not  escape  faster  than  ten  or  fifteen  drops  in  a  min- 
ute. It  is  best  after  once  starting  percolating  to  keep  a 
layer  of  menstruum  above  the  surface  of  the  drug  so  as  to 
prevent  the  access  of  air  to  its  interstices.  If  the  latter  hap- 
pens the  drug  contracts  into  irregular  masses  leaving  fur- 
rows and  fissures,  and  when  more  menstruum  is  added  it 
will  naturally  flow  through  these  channels  and  will  not  pene- 
trate the  drug  evenly. 

The  U.  S.  P.  gives  general  directions  for  percolation  and 
also  in  the  cases  of  the  individual  drugs  specifies  the  size  of 
the  powder  and  the  number  of  grams  to  be  used  together 
with  the  amount  and  character  of  the  menstruum.  It  also 
gives  directions  as  to  the  length  of  time  the  drug  is  to  mac- 
erate before  percolation. 

Percolation  is  one  of  the  most  important  processes  em- 
ployed in  pharmacy,  as  by  it  are  prepared  extracts  (page 
147);  fluid  extracts  (page  96);  and  tinctures  (page  168), 
and  it  will  be  used  in  the  manufacture  of  these  preparations 
in  the  following  pages.  In  addition  it  is  one  of  the  steps 
employed  in  the  isolation  of  alkaloids. 


.EXPERIMENTAL  PHARMACOLOGY  49 


50  LABORATORY  GUIDE  IN 


EXPERIMENTAL  PHARMACOLOGY  51 

Powders. 

A  very  considerable  group  of  medicinal  substances  is 
prescribed  in  powder  form.  Some  substances  act  better 
when  given  in  this  manner  and  certain  individuals  also 
find  them  easier  to  swallow  than  either  pills  or  capsules. 
To  be  best  suited  for  this  form  of  medication  the  drug 
should  be  fairly  insoluble  so  as  to  avoid  taste  and  should  not 
be  given  in  too  large  a  dose. 

The  manner  in  which  powders  are  dispensed  may  be 
learned  best  by  filling  a  prescription  such  as  the  following 
in  which  100  mg.  of  calomel  are  to  be  divided  into  eight 
powders.  On  account  of  the  weight  of  the  calomel  the  bulk 
of  each  powder  has  to  be  increased  by  another  powder 
which  is  inactive ;  in  this  case,  sugar  of  milk  being  used. 

R 

Hydrargyri  chloridi  mitis  o.i 
Sacchari  lactis  1.5 

Misce  et  divide  in  pulveres  No.  VIII. 
Sig. 

Take  one  powder  every  20  minutes  until  all  are  taken. 

Weigh  out  both  ingredients  and  put  the  calomel  into  a 
small  mortar  and  add  the  sugar  of  milk,  little  by  little,  mix- 
ing it  all  the  time  with  the  pestle,  using  only  light  pressure 
to  avoid  the  formation  of  compact  layers.  From  time  to 
time,  scrape  the  powder  together  into  the  middle  of  the 
mortar.  When  the  two  ingredients  are  thoroughly  mixed, 
empty  the  powder  onto  the  pill  tile  and  without  compress- 
ing it  at  all  arrange  it  in  an  oblong  parallelogram,  the  longer 
edge  being  near  the  scale  on  the  tile.     See  that  the  powder 


52 


LABORATORY  GUIDE  IN 


is  of  a  uniform  depth  over  the  whole  mass  and  if  it  is  not, 
scrape  some  powder  very  lightly  from  the  high  points  down 
to  the  thinner  parts.  The  powder  can  now  be  divided  easily 
into  eight  equal  portions  by  using  the  spatula  with  reference 
to  the  scale  on  the  tile.  The  powders  are  now  ready  to  be 
put  in  papers  which  are  obtained  by  the  dispenser  all  ready 
for  use. 

Take  eight  papers  and  make  a  narrow  fold  in  each  along 
one  of  the  longer  sides.  Put  a  powder  in  the  center  of  one 
cf  the  papers  and  bring  the  uncreased  edge  of  the  paper 
over  into  the  fold  previously  made  and  then  both  of  these 
are  brought  over  to  the  center,  creasing  the  whole  paper 
along  the  lines  formed.  The  ends  of  the  paper  are  now 
creased  by  pressing  them  over  the  sides  of  a  powder  box 
and  one  end  is  tucked  within  the  fold  of  the  other.  Fix 
all  the  powders  in  the  same  way  and  place  all  in  the  box, 
labeling  it  properly. 


nXPERlMUNTAL  PHARMACOLOGY  53 


54 


LABORATORY  GUIDE  IN 


MXPBRIMBNTAL  PHARMACOLOGY  55 

Capsules. 

Capsules  are  shells  of  gelatine  into  which  medicinal  sub- 
stances are  put  in  order  to  cover  up  the  taste.  The  sub- 
stances placed  in  the  capsules  may  be  either  in  solid  or  liquid 
form.  Empty  capsules  made  of  gelatine  are  purchased  by 
the  pharmacist  so  that  the  problem  is  merely  one  of  filling. 
The  process  can  be  best  learned  by  the  experience  gained  in 
filling  one  or  two  prescriptions. 
R 

Quininas  Sulphatis  i.o 

Pone  in  capsulas  No.  VIII. 
Sig. 

Take  one  capsule  every  three  hours. 

To  fill  this  prescription  the  quinine  sulphate  is  weighed 
out  and  placed  upon  the  pill  tile  and  spread  out  into  an 
oblong  parallelogram  with  the  powder  of  uniform  depth. 
This  mass  of  powder  is  now  divided  lengthwise  with  the 
spatula  and  then  crosswise  in  the  middle  and  then  on  eacli 
side  of  the  middle,  thus  making  eight  equal  piles  of  quinine. 
The  large  end  or  bottom  part  of  the  capsule  (size  No.  2) 
is  now  pressed  down  on  the  powder  repeatedly  until  all  the 
powder  in  one  of  the  piles  is  packed  into  it  and  then  the  top 
part  is  placed  on  the  base,  thus  closing  it.  The  process  is 
repeated  with  the  other  seven  capsules  until  all  are  filled. 
All  traces  of  quinine  must  be  removed  from  the  outside  of 
the  capsules  either  by  wiping  carefully  with  a  clean  soft 
cloth  or  washing  them  in  alcohol  and  then  drying  with  the 
cloth. 

There  are,  of  course,  various  modifications  of  this  method 
of  filling  capsules,  made  necessary  by  the  different  char- 


56  LABORATORY  GUIDE  IN 

acteristics  of  the  individual  powders  which  it  may  be  de- 
sired to  place  in  the  capsule.  For  example,  some  powders 
lack  adhesiveness  and  would  run  out  of  the  shell  each  time 
it  was  raised.  Such  powders  may  be  put  in  by  means  of 
the  spatula  or  by  the  use  of  some  form  of  filling  apparatus 
--many  of  which  are  on  the  market. 

Oily  or  alcoholic  liquids  may  also  be  dispensed  in  hard 
capsules  as  for  example  in  the  following  prescription : 

R 

Olei  Terebinthinge  4. 

Pone  in  capsulas  No.  VIII. 

For  this  purpose  select  eight  empty  capsules  (size  No.  o) 
and  remove  the  tops,  placing  them  mouth  downwards  upon 
some  moist  filter  paper.  This  will  soften  the  gelatine  at 
the  point  of  contact  with  the  moisture.  Draw  up  some  of 
the  oil  of  turpentine  into  a  one  or  two  cubic  centimeter 
graduated  pipette  and  taking  the  bottom  part  of  the  cap- 
sule between  the  thumb  and  first  finger  of  the  left  hand, 
allow  0.5  cc.  of  the  oil  to  flow  into  the  capsule,  taking  care 
none  gets  on  the  outside.  Now  replace  the  top,  thus  clos- 
ing the  capsule,  and  rotate  it  between  the  thumb  and  finger 
to  seal  it  by  compressing  the  soften  ring  of  gelatine  which 
was  at  the  mouth  of  the  top  portion.  Repeat  the  process 
with  the  other  capsules. 

Soft  or  elastic  gelatine  capsules  are  also  employed  in 
the  dispensing  of  oily  liquids. 


BXPBRIMBNTAL  PHARMACOLOGY 


57 


58  LABORATORY  GUIDE  IN 


BXPBRIMBNTAL  PHARMACOLOGY  59 

Cachets. 

A  convenient  mode  of  prescribing  powders,  especially 
those  which  have  a  disagreeable  taste  and  are  not  to  be 
given  in  too  large  doses,  is  by  enclosing  them  in  cachets 
which  are  small  concave  discs  made  of  flour.  The  powder 
is  put  between  two  of  the  discs  and  the  edges  then  sealed 
forming  a  tight  compartment. 

Fill  the  following  prescription: — 

R 

Quininge  'Sulphatis  0.5 

Pone  in  cachetas  No.  III. 
Sig. 

One  cachet  every  three  hours. 

Weigh  out  the  qtiinine  sulphate  and  placing  it  on  a  pill 
tile  divide  it  into  three  equal  portions.  Holding  one  of 
the  paste  discs  in  the  left  hand,  by  means  of  the  spatula 
place  one  of  the  powders  of  quinine  in  the  middle  of  the 
disc,  taking  care  none  of  the  quinine  gets  on  the  edge 
of  the  disc.  Moisten  the  edge  of  another  disc  with  a  very 
httle  water,  and  then  place  it  against  the  edge  of  the  disc 
which  holds  the  quinine.  Press  the  two  edges  together 
firmly,  thus  sealing  the  cachet.  Fill  the  other  cachets  in 
the  same  manner  and  remove  any  quinine  which  may  be 
on  the  outside.  These  cachets  may  be  swallowed  very 
easily  if  they  are  first  softened  by  being  placed  in  water 
for  a  few  moments.  ' 


6o  LABORATORY  GUIDE  IN 

Pills. 

Pills  are  spherical  or  ovoid  masses  of  medicinal  sub- 
stances of  a  size  convenient  for  swallowing.  In  weight  they 
should  not  exceed  0.3  G.,  the  average  being  about  0.2  G. 

This  method  of  administering  solid  medicinal  substances 
is  perhaps  more  common  than  any  other,  partly  on  account 
of  convenience  arid  also  because  substances  which  have  a 
disagreeable  taste  may  be  given  most  agreeably  in  this  way. 
Drugs  which  are  corrosive  or  are  very  deliquescent  or 
which  have  to  be  given  in  very  large  doses  should  not  be 
given  in  pills.  Those  which  are  best  suited  for  administra- 
tion in  this  form  are  powders  and  extracts. 

The  first  step  in  pill  making  is  to  form  the  pill  mass, 
which  should  be  of  doughy  consistency  but  not  sticky.  The 
mass  consists  of  two  parts ;  the  active  ingredients  which 
are  to  be  incorporated  in  the  pill  and  some  substance  (the 
excipient)  which  will  give  the  proper  degree  of  consistency 
and  adhesiveness.  The  excipient  will  vary  with  the  char- 
acter of  the  active  ingredients.  Some  of  the  powdered  ex- 
tracts only  require  water  to  give  them  the  proper  consis- 
tency, while  dilute  alcohol  may  be  needed  in  other  extracts. 
Some  fluid  preparations  may  be  made  into  pills  by  using  an 
inert  powder  and  on  the  other  hand  a  non-adhesive  powder 
ma)''  be  made  into  a  pill  mass  by  the  use  of  soft  extracts 
or  by  the  use  of  such  an  excipient  as  a  combination  of 
glycerin  and  tragacanth  (glycerite  of  tragacanth).  The 
choice  of  an  excipient  is  very  important,  as  it  largely  de- 
termines the  usefulness  of  pills  if  they  are  kept  for  some 
time.     P'or  example,  a  pill  made  with  acacia  as  excipient 


EXPERIMENTAL  PHARMACOLOGY  6i 


62  LABORATORY  GUIDE  IN 


EXPERIMENTAL  PHARMACOLOGY  63 

may  become  so  hard  that  it  will  pass  through  the  body  un- 
changed, while  with  other  excipients  the  pill  may  dry  out 
and  crumble  to  pieces  after  they  have  been  made  for  a 
short  time.  The  formation  of  a  pill  mass  may  be  learned 
best  by  filling  the  following  prescription,  which  calls  for 
ten  pills  to  be  made  from  a  gram  of  extract  of  gentian. 

R       Extracti  Gentianse,   i. 
Divide  in  pilulas,  No.  X. 

Counterpoise  two  slips  of  paper  on  the  scale  pans  and 
weigh  out. the  correct  amount  of  extract,  which  is-  then 
transferred  to  a  mortar.  (If  some  of  the  extract  sticks  to 
the  piece  of  paper  moisten  the  opposite  side  of  the  paper 
with  water  and  wait  a  few  moments,  after  which  the  re- 
mains of  the  extract  may  be  easily  scraped  off.) 

Make  the  extract  into  a  pill  mass  of  the  character  de- 
scribed above.  If  it  is  too  hard  add  a  little  water  or  dilute 
alcohol;  if  too  soft  add  a  small  quantity  of  a  powder,  such 
as  starch  or  the  powdered  extract  of  liquorice.  Triturate 
the  mass  thoroughly  in  the  mortar  (not  in  the  hands!)  so 
as  to  get  the  two  substances  uniformly  distributed  through 
each  other.  When  the  mass  is  completed,  work  it  up  with 
the  hands  into  a  short  cylinder  and  transfer  it  to  the  pill 
slab,  over  which  has  been  scattered  a  small  quantity  of  a 
dusting  powder,  such  as  lycopodium.  Roll  out  the  mass 
with  the  wooden  roller  into  an  even  cylinder  of  the  right 
length  to  correspond  with  the  scale  on  the  slab  (in  this  case 
roll  out  to  10),  taking  care  that  the  ends  of  the  cylindrical 
mass  are  ''squared  up."  With  the  cylinder  over  the  scale, 
cut  it  with  the  spatula  into  the  required  number  of  pieces, 
taking  care  that  the  spatula  is  always  held  at  right  angles  to 


64  LABORATORY  GUIDE  IN 

the  mass.  The  Httle  sections  of  the  pill  mass  are  now  to  be 
moulded  with  the  thumb  and  fingers  into  spherical  shape 
and  then  placed  in  the  pill  box  with  a  small  amount  of  ly co- 
podium  and  shaken  about  so  that  they  may  be  completely 
covered  by  powder  which  will  prevent  them  sticking  to- 
gether.    Discard  the  excess  of  powder. 

To  mask  the  taste  of  the  pills  they  are  usually  coated 
with  some  substance  such  as  gelatin,  sugar,  chocolate,  etc. 

Examine  the  pills  after  a  day  or  two  and  see  if  they  have 
retained  their  shape  or  if  they  have  flattened  out.  Weigh 
five  pills  and  see  how  nearly  they  correspond  in  weight. 


EXPERIMENTAL  PHARMACOLOGY 


65 


66  LABORATORY  GUIDE  IN 


■  MXPBRIMBNTAL  PHARMACOLOGY  67 

Emulsions. 

Emulsions  are  aqueous  preparations  in  which  oils,  resins 
or  insoluble  powders  are  suspended  by  means  of  gummy 
substances    (emulsifiers). 

The  object  of  emulsification  is  to  render  substances  in- 
soluble in  water  freely  miscible  in  that  fluid,  so  that  any 
disagreeable  taste  may  be  more  easily  disguised  and  the 
absorption  of  the  oil  rendered  more  easy.  In  nature,  some 
emulsions  exist  already  formed,  as  for  example,  milk,  the 
juice  of  plants,  the  yolk  of  eggs,  etc. 

There  are  a  large  number  of  substances  which  can  be 
used  as  emulsifiers,  the  most  important  being  acacia.  Con- 
densed milk  and  yolk  of  egg  are  superior  to  acacia  for  this 
purpose,  but  possess  the  disadvantage  of  becoming  stale. 

In  some  substances,  as  for  instance  in  gum  resins,  the 
gum  needed  to  form  an  emulsion  is  found  present  with  the 
resin.  In  the  seeds  of  some  plants  albuminous  substances 
are  found  and  these  act  as  emulsifiers  of  the  oils  present 
in  the  seed,  rendering  the  addition  of  gums  unnecessary. 

The  various  types  of  emulsions  are  prepared  according 
to  the  following  directions : 

I.     Seed  emulsion  prepared  from  bitter  almonds. 

Take  four  or  five  bitter  almonds  and  remove  their  skins. 
This  may  be  done  in  two  ways.  They  may  be  dropped  for 
a  few  moments  in  very  hot  water  and  then  in  cold,  when 
the  skins  can  be  slipped  off.  The  danger  with  this  method 
is  that  the  boiling  water  may  destroy  the  ferment,  emulsin, 
which  aids  in  the  emulsification.     To  avoid  this,  the  seeds 


68  LABORATORY  GUIDE  IN 

may  be  soaked  in  lukewarm  water  for  half  an  hour,  or  until 
their  skins  are  loosened. 

Break  up  the  seeds  and  put  them  in  a  clean  mortar  and 
add  a  small  quantity  of  water  (one  part  to  ten  of  seeds) 
and  with  the  pestle  rub  the  seeds  up  into  a  thick,  creamy 
paste  which  shall  be  free  from  lumps.  More  water  can  be 
added  now,  gradually,  until  a  milky  solution  is  formed, 
which  may  be  strained  if  necessary. 

II.  Emulsion  of  a  gum  resin. 

Prepare  lOO  cc.  of  the  emulsion. of  asafoetida  according 
to  the  U.  S.  P. 

III.  Emulsion  of  a  fixed  oil. 

Among  the  various  methods  employed  in  making  emul- 
sions, that  known  as  the  Continental  is  probably  the  most 
satisfactory,  and  is  to  be  used  in  the  following  experiment. 

Prepare  60  cc.  of  a  50%  emulsion  of  castor  oil.. 

In  this  method  a  primary  emulsion  or  nucleus  is  first 
formed  with  certain  proportions  of  the  ingredients  and  this 
riucleus  can  then  be  diluted  with  water  without  the  fear 
that  separation  will  occur. 

The  proportion  by  weight  is  as  follows : 

Oil ' 4 

Water   2 

Gum    (powdered  acacia)    i 

Using  this  formula,  calculate  the  amount  of  each  ingredi- 
ent needed  and  weigh  them  out  carefully  in  clean  vessels. 
Pour  the  oil  into  a  clean,  dry  mortar  and  add  the  acacia,  and 
with  the  pestle  stir  the  two  together  rapidly  until  a  uniform 
mixture  is  obtained.     (This  must  be  done  quickly,  as  other- 


UXPBRIMBNTAL  PHARMACOLOGY  69 


yo  LABORATORY  GUIDE  IN 


■BXPBRIMUNTAL  PHARMACOLOGY  71 

wise  the  oil  and  acacia  remain  in  contact  too  long  and  the 
latter  tends  to  become  hard  and  insoluble.  The  object  is 
merely  to  get  each  particle  of  gum  incased  in  oil.)  To  the 
oil-acacia  mixture  add  all  the  water  (as  determined  by  the 
above  formula)  and  triturate  the  mixture  rapidly  until  it 
becomes  a  thick,  white,  creamy  emulsion.  In  the  trituration, 
which  should  take  from  3  to  5  minutes,  no  pressure  is 
needed,  but  a  rapid  motion  is  very  important.  The  pestle 
should  be  held  loosely  between  the  thumb  and  first  two  or 
three  fingers  and  the  motion  given  not  only  from  the  shoul- 
der and  elbow,  but  also  from  the  wrist  and  fingers.  It  is 
best  to  move  the  pestle  always  in  one  direction,  but  it  is  not 
essential  to  do  so. 

When  the  nucleus  is  complete  it  can  be  poured  into  a 
bottle  and  water  added  to  make  up  the  required  bulk.  It 
should  then  be  well  shaken. 

Such  an  emulsion  may  separate  after  some  hours  into  two 
layers  without  showing  any  separated  oil.  Shaking  will  re- 
store the  uniform  appearance.  Such  separation  is  entirely 
analogous  to  that  which  occurs  in  milk  when  the  cream 
rises  to  the  surface. 

Emulsions  can  be  flavored  by  various  agencies,  such  as 
the  volatile  oils,  but,  as  a  general  rule,  some  of  the  less 
powerful  flavors  will  disguise  the  taste  of  the  oil  better,  as 
for  instance,  liquorice  extract,  coffee,  vanilla,  or  chocolate. 
They  may  be  sweetened  by  small  quantities  of  sugar  or 
saccharin. 

Acids,  alcohol,  or  glycerin,  if  added  in  any  except  the 
smallest  quantities,  cause  the  emulsion  to  separate  (break). 


72  LABORATORY  GUIDE  IN 

Ointments. 

Ointments  are  soft  fatty  preparations  which  are  in- 
tended for  appHcation  to  the  skin.  The  purpose  of  an 
ointment  may  be  merely  to  act  as  a  protective  to  the  skin 
or  to  render  it  softer  and  more  elastic,  in  either  case  the 
action  being  merely  a  local  one.  Then,  too,  active  sub- 
stances are  sometimes  incorporated  into  ointments  in  order 
that  they  may  be  absorbed  and  exert  a  general  or  systemic 
action  upon  the  body. 

In  order  that  ointments  may  be  used  for  these  different 
purposes,  it  is  evident  that  substances  possessing  different 
properties  must  be  used  in  their  formation.  In  one  in- 
stance it  is  not  desirable  for  the  ointment  to  be  absorbed, 
whereas  in  another  case  lack  of  penetrating  ability  might 
be  disadvantageous.  To  give  these  different  properties  to 
ointments,  there  are  three  substances  used  most  commonly 
in  their  formation;  namely,  vaseline,  lard  (or  lard  con- 
taining benzoin  to  keep  it  from  becoming  rancid),  and 
hydrous  wool  fat  or  lanolin.  Vaseline  is  the  most  un- 
changeable of  the  bases,  being  unaffected  by  air,  moisture 
or  chemicals.  However,  it  is  not  absorbed  well,  nor  does 
it  give  up  readily  the  medicinal  agents  incorporated  in  it, 
so  that  it  is  best  suited  for  ointments  designed  for  pro- 
tective purposes  only.  Benzoinated  lard  is  very  valuable 
as  a  base  as  it  will  take  up  about  fifteen  per  cent  of  water 
without  losing  its  ointment-like  consistency  and  in  addi- 
tion it  readily  yields  up  for  absorption  any  medicinal  sub- 
stances which  have  been  incorporated  in  it. 

Wool  fat  is  also  largely  used  as  it  will  take  up  a  very 
considerable  amount  of  water — even  an  equal  weight — and 


T^XPBRIMUNTAL  PHARMACOLOGY  'j2> 


^4  LABORATORY  GUIDE  IN 


UXPBRIMBNTAL  PHARMACOLOGY  75 

in  addition  it  is  easily  absorbed.  Its  chief  disadvantages  are 
that  it  is  not  so  easily  worked  as  other  fats  and  that  it  is 
quite  sticky.  These  imdesirable  features  may  be  lessened  by 
incorporating  other  fats  with  it  in  different  proportions. 

Prepare  thirty  grams  of  belladonna  ointment  (Unguentum 
Belladonna)  according  to  the  directions  given  in  the  U.  S. 
Pharmacopoeia. 

In  preparing  this  ointment  note  especially  the  physical 
characteristics  of  the  lard  and  the  wool  fat. 

It  is  hardly  necessary. to  say  that  the  finished  ointment 
should  be  perfectly  homogeneous  and  free  from  any  gritty 
particles. 

Examine  a  specimen  of  vaseline  (Petrolatum).  Look 
over  also  the  composition  of  other  ointments  than  the  bella- 
donna ointment  as  they  are  described  in  the  Pharmacopoeia. 


76  LABORATORY  GUIDB  IN 

Suppositories. 

Suppositories  are  solid  medicated  substances  usually  of 
a  conical  form  and  designed  for  introduction  into  the  vari- 
ous passages  of  the  body  where  they  will  become  liquid 
and  exercise  a  local  action  or  else  be  absorbed  and  have 
a  general  or  systemic  effect.  They  consist  essentially, 
therefore,  of  some  drug  and  a  base  with  which  the  medicinal 
substance  is  incorporated  and  which  is  solid  at  ordinary 
temperatures,  but  which  liquifies '  slightly  below  the  body 
temperature. 

The  base  most  commonly  employed  is  cacao  or  cocoa  but- 
ter (Oleum  Theobromatis)  which  is  obtained  from  the 
seeds  of  the  plant  Theobroma  cacao,  from  which  chocolate 
is  obtained.  The  melting  point  of  this  substance  lies  be- 
tween 86°  and  95°  F.  Other  bases  which  are  used  for  spe- 
cial purposes  are  glycerin-gelatin  and  sodium  stearate. 

The  size  and  shape  of  suppositories  will  depend  upon  the 
base  employed  in  their  manufacture,  and  the  use  to  which 
they  are  to  be  put.  If  they  are  to  be  made  from  the  oil 
of  theobroma,  the  cone  shaped  rectal  suppositories  should 
weigh  about  two  grammes ;  the  pencial  shaped  suppository 
for  the  urethra  about  the  same  amount,  and  the  globular 
suppositories  for  the  vagina,  four  grammes.  If  on  the 
other  hand,  they  are  made  with  glycerin-gelatine  as  a  base, 
the  weights  above  given  must  be  doubled. 

There  are  three  ways  of  making  suppositories  using  the 
oil  of  theobroma  as  a  base. 

In  the  fusion  process,  the  oil  is  gently  melted  and  the 
medicinal  substance  incorporated  with  it  while  it  is  still 


BXPBRIMENTAL  PHARMACOLOGY  77 


78  LABORATORY  GUIDE  IN 


EXPERIMENTAL  PHARMACOLOGY  79 

warm  and  as  the  mass  is  cooling  it  is  poured  into  well  cooled 
moulds  and  allowed  to  harden. 

In  the  cold  compression  method,  the  mixed  drug  and 
base  are  placed  in  a  special  apparatus  and  under  pressure 
moulded  into  the  required  shape.  Complete  directions  for 
these  methods  are  to  be  found  in  the  U.  S.  Pharmacopoeia. 

The  third  method,  and  the  one  usually  employed  when 
only  a  few  are  to  be  made  is  carried  out  in  the  following 
manner,  which  may  be  illustrated  by  filling  a  prescription 
calling  for  three  suppositories. 

R 

Acidi  Tannici  0.3 

Olei  Theobromatis  6. 

Fiant  suppositoria  No.  iii. 

Sig. 

Use  as  directed. 

These  three  suppositories  are  to  be  made  by  hand  as 
follows.  Put  the  tannic  acid  in  a  mortar  and  add  about 
an  equal  weight  of  the  oil,  which  has  been  grated.  Mix 
the  two  thoroughly  and  then  add  the  remainder  of  the 
grated  oil,  working  the  mass  in  the  mortar  until  it  be- 
comes homogeneous  and  plastic.  If  necessary  to  make  the 
mass  cohesive,  about  three  drops  of  oil  of  sweet  almond  or 
of  glycerin  may  be  added.  Transfer  the  mass  to  the  pill 
tile  and  form  it  into  a  cylinder  of  convenient  length  and 
cut  it  into  three  equal  parts.  Form  each  piece  now  into  a 
conical  suppository  by  means  of  the  fingers  and  spatula. 
They  should  all  be  of  the  same  size  and  shape.  Dust  them 
with  a  little  lycopodium  or  starch  to  keep  them  from  stick- 
ing together  in  the  box. 


8q  laboratory  guide  in 

Physiology  of  the  Nervous  System  of  the  Frog. 

In  order  to  study  the  action  of  a  drug  upon  the  nervous 
system  of  a  frog  it  need  hardly  be  pointed  out  that  it  is  of 
prime  importance  to  understand  the  physiology  of  the  differ- 
ent parts  of  that  system.  To  illustrate,  a  certain  drug  will 
depress  the  cerebrum,  while  a  second  drug  will  stimulate  the 
spinal  cord  and  a  third  may  paralyze  the  peripheral  nerves ; 
now,  only  by  knowing  the  normal  functions  of  these  di- 
visions of  the  nervous  system  can  we  recognize  and  interpret 
the  symptoms  arising  from  an  increase  or  diminution  in  their 
activity,  and  thereby  locate  the  point  of  action  of  the  drug 
employed. 

First,  examine  the  normal  frog,  observing  its  spontaneous 
movements  and  the  method  of  leaping  and  what  degree  of 
irritation  is  necessary  to  make  it  move.  Note  also  its  co- 
ordination of  the  muscles  in  swimming;  its  recovery  of  bal- 
ance when  slowly  turned  on  a  sloping  board,  its  recovery 
v/hen  placed  on  its  back,  its  reflexes  when  the  toe  is  pinched, 
the  croaking  when  its  back  and  sides  are  stroked,  the  rate  of 
respiration  and  heart  beat.  (The  latter  can  usually  be 
counted  when  the  animal  is  turned  on  its  back  and  placed  in 
a  good  light,  a  slight  shadowy  movement  of  the  skin  being 
seen  in  the  cardiac  region  with  each  pulsation  of  the  organ.) 

Having  studied  the  intact  animal,  separate  the  cerebral 
hemispheres  from  the  optic  lobes  by  thrusting  the  point  of 
a  scalpel  through  the  roof  of  the  skull  on  a  line  joining  the 
posterior  borders  of  the  eyelids,  gently  rocking  the  scalpel 
from  side  to  side  so  as  to  completely  sever  the  hemispheres 
from  their  inferior  connections.    As  will  be  seen  from  the 


-BXPERIMENTAL  PHARMACOLOGY  8i 


82  LABORATORY  GUIDE  IN 


EXPBRIMENTAL  PHARMACOLOGY 


83 


accompanying  figure,  ;:uch  a  cut 
will  also  destroy  the  optic  thalami 
and  some  symptoms  will  result 
from  such  destruction,  but  for  all 
purposes  here,  the  differences  in 
results  obtained  are  not  important. 
After  allowing  the  animal  a  few 
minutes  to  recover  from  the  effects 
of  the  shock  of  the  operation 
(which  must  be  done  in  all  cases) 
compare  its  condition  with  that  of 
the  normal  animal. 

Make  full  notes  of  all  changes 
seen. 

Next  remove  the  optic  lobes 
from  the  same  animal  by  cutting 
as  before  with  the  point  of  a 
scalpel  on  a  line  joining  the  cen- 
ters of  the  tympanic  membranes. 
These  membranes  are  seen  on  the  sides  of  the  head  posterior 
to  the  eyes  as  two  fiat  circular  surfaces  about  3mm.  in 
diameter.  Wait  a  short  time  as  before  and  then  study  the 
effects  of  the  operation.  Hang  up  the  animal  by  means  of 
a  wire  hook  passed  through  the  lower  jaw  and  observe  the 
reflexes  on  pinching  the  toes  or  on  dipping  the  toe  in  very 
dilute  acid  (made  by  adding  dilute  sulphuric  acid  to  water). 
Wash  off  the  acid  with  water. 

Using  the  same  frog  thrust  a  fine  wire  down  the  spinal 
canal  so  as  to  destroy  the  cord.  Observe  any  change  in  the 
reflexes.    What  is  the  condition  of  the  heart  and  respiration  ? 


Fig.  No.  4.  Frog's  Brain,  a. 
Cerebral  hemispheres;  b.  Optic 
thalami;  c.  Optic  lobes.  d. 
Cerebellum,  e.  Medulla.  (Stein- 
er.) 


84  LABORATORY  GUIDE  IN 

Examine  the  activity  of  the  peripheral  nerves  in  the  fol- 
lowing manner ;  hold  the  frog  back  upwards  in  the  hand  and 
apply  the  electrodes  from  the  induction  coil  (tetanizing  cur- 
rent) to  the  lumbar  spine.  Observe  how  long  the  muscles 
will  stay  contracted  before  fatigue  sets  in. 

Dissect  out  the  sciatic  nerve  by  an  incision  along  the  pos- 
terior surface  of  the  thigh  and  stimulate  it  with  the  electric 
current  as  before. 

Lay  a  few  crystals  of  sodium  chloride  on  the  nerve  and 
moisten  them  with  a  drop  of  water.  Observe  any  change 
in  the  muscles.    Explain. 

Expose  the  gastrocnemius  muscle  and  stimulate  it  directly 
with  the  electric  current  and  also  with  salt  crystals. 

Describe  the  functions  of  the  different  parts  of  the  nerv- 
ous system  in  the  frog  as  learned  from  the  symptoms  arising 
from  their  removal. 

Action  of  Acid  Puchsin. 

In  connection  with  the  study  of  the  physiology  of  the 
nervous  system  of  the  frog  the  action  of  acid  fuchsin  is 
very  interesting  and  instructive.  This  drug  when  injected 
into  frogs  in  proper  dosage  apparently  produces  no  effects 
for  from  12  to  24  hours.  If,  however,  it  is  given  in  the 
same-sized  dose  to  an  animal  suffering  from  fatigue  or  to 
one  in  which  the  cerebral  hemispheres  have  been  injured  the 
symptoms  will  appear  in  about  fifteen  minutes. 

Experiment  i.  Using  a  1.5%  solution  of  acid  fuchsin 
inject  into  the  anterior  lymph  sac  (see  page  13)  of  a  frog 
0.3  mg.  acid  fushsin  per  gram  body  weight  of  animal.  Re- 
place the  frog  under  the  bell  jar  and  study  it  from  time  to 
time,   observing   general   activity,   reflexes,    etc.      After    10 


■UXPBRIMBNTAL  PHARMACOLOGY  85 


86  LABORATORY  GUIDE  IN 


■EXPERIMENTAL  PHARMACOLOGY  87 

or  15  minutes  sever  the  cerebral  hemispheres  from  the  rest 
of  the  brain,  according  to  the  method  described  earher, 
and  replace  the  frog  on  the  table  for  observation.  If  con- 
vulsions appear  study  them  carefully,  making  adequate  notes 
and  then  try  to  locate  the  point  of  action  of  the  poison  by 
cutting  off  the  optic  lobes,  destroying  the  medulla  and  cord 
noting  changes  that  occur  after  each  operation. 

Where  does  the  drug  act  to  produce  the  convulsions  and 
v/hat  light  does  the  experiment  throw  upon  the  function 
of  the  cerebrum? 


88  LABORATORY  GUIDE  IN 

Soporifics  or  Hypnotics. 

Soporifics  or  Hypnotics  are  drugs  which  are  used  to  pro- 
duce sleep.  They  comprise  a  very  large  and  important 
group  of  substances  which  are  extensively  employed  in  medi- 
cine. In  their  action  they  are  closely  related  to  chloroform 
and  ether,  but  they  are  not  used  to  produce  anaesthesia  in 
man  owing  to  the  fact  that  it  is  impossible  to  regulate  suf- 
ficiently accurately  the  depth  of  the  anaesthesia  induced  by 
them. 

Chloral  (chloral  hydrate),  the  most  important  member  of 
the  series,  was  introduced  into  medicine  by  Liebriech  in 
1S69,  and  as  a  pure  hypnotic  drug  is  probably  the  most  re- 
liable in  use  today.  The  other  members  of  the  group  which 
are  of  greatest  importance  are  Sulphonal  (Sulphonmeth- 
ane),  Paraldehyde  and  Veronal. 

Examine  each  drug  as  to  appearance,  odor  and  taste. 
.     I.     Examine  the  solubility  of  the  following  soporifics : 


Chloral 


Sulphonal. 


Paraldehyde. 


Veronal. 


Cold   water. 
Hot  water. 
Alcohol. 
Ether. 


Wrap  up  a  few  crystals  of  chloral  in  paper,  set  aside  and 
examine  them  after  a  few  days. 

In  what  ways  would  you  think  these  drugs  could  best  be 
prescribed  in  practice? 

II.  Put  a  few  crystals  of  chloral  in  a  test  tube  and  add 
a  small  amount  of  potassium  hydrate  solution  and  heat 
gently.     Note  the  odor  of  the  fumes.    What  is  formed? 


liXPERIMBNTAL  PHARMACOLOGY  89 


go  LABORATORY  GUIDE  IN 


nXPERIMBNTAL  PHARMACOLOGY  91 

CCl3-CH0-H,0  +  KOH  =  H-COOK  +  ? 
Chloral  hydrate.  Potassium  formate. 

III.  Select  three  frogs  of  about  equal  size  and  mark 
each  frog  in  some  way  so  that  it  can  be  recognized.  (This 
is  probably  done  most  easily  by  making  a  drawing  showing 
the  outlines  of  the  marking  on  the  back.)  Examine  the 
frogs,  making  a  record  of  the  heart  rate  and  respiration  in 
each. 

Inject  into  the  anterior  lymph  sac  (page  13)  of  one  frog 
5  mg.  of  chloral  dissolved  in  water.  After  studying  this 
animal  for  about  10  minutes  inject  into  the  second  frog  2vT 
mg.  of  chloral.  In  like  manner  after  observing  this  animial 
for  a  time  and  making  notes  inject  into  the  third  frog  50 
mg.  of  chloral.  These  amounts  may  be  obtained  most  ac- 
curately by  making  5  or  10  cc.  of  a  5%  solution  and  then 
diluting  small  quantities  (i  cc.)  so  as  to  get  the  required 
doses. 

Make  a  record  of  the  time  when  each  injection  is  made. 
After  injection  replace  the  animals  under  the  bell  jar  and 
observe  them  frequently  (especially  those  with  the  larger 
doses)  as  to  spontaneous  movements,  co-ordination,  reflexes, 
respiration,  heart  beat,  etc. 

Keep  a  careful  record  of  the  condition  of  each  frog  at 
each  examination. 

This  can  be  done  most  systematically,   not   only  in  this   experiment,   but   also 
in  those  to  follow,  by  keeping  a  record   of  the  time   when   each  examination  is 
made  and  the  conditions  found.     By  doing  this  it  also   gives   some  idea  of  the 
length  of  time  it  takes  the  drugs  to  act  when  given  in  diiferent  sized  doses. 
Such  a   record  would  start   something  as   follows: 
Feb.    10,  '15 — Frog.     2^   G. 

2:30 — Heart  56,   respiration   30   per  minute. 

2:32 — Injected   20   mg.    chloral   in   water   into   anterior   lymph   sac. 
2:3s — Frog  quiet,  apparently   normal;   reflexes,   normal;   heart,   54;   respir- 
ation,   28. 
2:40 — Frog    jumps    if    urged    strongly,    does    not    regain    sitting    position 
easily    when    it    alights;     has    difficulty    in    turning    over    when 
placed  on  back.     Reflexes  lessened;  heart,  40;  respiration,  15,  etc. 


92  LABORATORY  GUIDB  IN 

Such  a  record  should  give  an  accurate  picture  of  the  con- 
dition of  the  animal  at  each  stage  of  the  drug's  action. 

If  complete  paralysis  occurs  in  any  animal  determine 
whether  the  action  is  on  the  central  nervous  system  or  is 
peripheral. 

This  is  done  by  stimulating  over  the  lumbar  spine  with  the  electric  current 
and  if  the  muscles  of  the  leg  contract  the  paralysis  must  be  central;  if  they 
do  not  contract  see  whether  the  trouble  is  in  the  nerve  or  in  the  muscles  by 
stimulating  the  latter  directly  with  the  current. 

Also  in  one  of  the  animals  thus  paralyzed,  expose  the 
heart  (page  14)  and  examine  and  record  its  condition. 

The  frog  given  50  mg.  may  be  used  for  this  purpose.    The 
others  may  be  replaced  at  the  end  of  the  day's  work  under 
the  bell  jar  and  moistened  and  examined  on  the  following, 
day.     Some  will  doubtless  recover.     Notes  should  be  kept 
of  their  progress. 

With  the  knowledge  gained  of  the  physiology  of  the 
nervous  system  study  the  records  of  the  three  frogs  and 
from  the  symptoms  recorded  determine  the  points  of  action 
of  the  drug  in  the  varying  doses. 

IV.  Inject  into  the  anterior  lymph  sac  of  a  frog  0.5  cc. 
of  a  10%  solution  of  paraldehyde  in  water.  Compare  the 
symptoms  shown  by  this  animal  with  those  manifested  by 
the  chloral  frogs  in  Exp.  III.  Pay  particular  attention  to  the 
condition  of  the  heart,  which  can  probably  be  sufficiently 
well  studied  (as  to  its  rate)  through  the  chest  wall  without 
exposing  it.    Keep  the  animal,  as  it  will  probably  recover. 

The  difference  in  action  of  the  two  drugs  upon  the  heart 
is  explained  by  the  difference  in  the  constitution  of  the  two 
molecules.  The  chloral  molecule  contains  chlorine  and  these 
chlorine  compounds  all  have  a  decided  action  on  the  heart 
muscle  which  is  absent  in  the  case  of  paraldehyde,  whose 


EXPERIMBNTAL  PHARMACOLOGY  93. 


94  LABORATORY  GUIDE  IN 


UXPBRIMBNTAL  PHARMACOLOGY  95 

molecule,    CgH^aOa,    contains   no   chlorine.      Compare   the 
action  of  chloroform  and  ether  on  the  heart  (page  187). 
Other  members  of  this  group  are,  Trional. 

Tetronal. 

Urethane. 

Chloretone. 

Chloralose,  etc. 

Therapeutics} 

The  chloral  group  of  drugs  depresses  the  central  nervous 
system.  For  this  purpose  it  would  be  used  in  cases  of  in- 
somnia and  of  nervous  excitement.  Chloral  and  its  allies  are 
very  valuable  also  in  delirium  and  in  various  forms  of  con- 
vulsions, as  for  instance,  those  due  to  poisoning  with  certain 
drugs  or  those  due  to  specific  diseases  (tetanus).  In  certain 
forms  of  insanity  (mania)  and  in  some  nervous  diseases 
with  increased  activity  of  the  brain  and  spinal  cord  (chorea 
or  St.  Vitus's  dance)  the  quieting  effects  of  these  drugs  are 
largely  made  use  of. 

On  account  of  its  action  on  the  heart  chloral  must  be  em- 
ployed with  care  in  persons  who  are  already  affected  with 
certain  forms  of  heart  disease  and  its  effect  on  this  organ 
must  also  be  watched  with  care  in  case  of  its  prolonged  use, 
or  better,  some  non-chlorine  containing  soporific  may  be 
used  in  its  stead. 


^  At  the  conclusion  of  the  study  of  a  drug  group  a  short  disctission  of 
the  therapeutic  uses  of  the  drugs  contained  therein  will  be  given.  Such  dis- 
cussions are  not  intended  to  give  compete  resume  of  all  the  therapeutic  uses 
of  the  drugs  studied,  but  only  those  will  be  mentioned  as  would  be  indicated 
by  the  results  obtained  in  the  foregoing  experiments.  The  student  will  thus  be 
shown  how  the  knowledge  gained  by  the  science  of  Pharmacology  is  trans- 
ferred to  and  made  use  of  in  the  Therapeutic  art;  or,  in  other  words,  in 
rational  medicine. 


96  LABORATORY  GUIDE  IN 

Nux  Vomica. 

Nux  Vomica,  the  seeds  of  Strychnos  nux  vomica,  an  East 
Indian  tree,  contains  two  alkaloids  upon  which  the  activity 
of  the  drug- depends.  By  far  the  more  important  of  the  two 
is  Strychnine  which  is  present  in  from  .9  to  2.  per  cent;  the 
second  alkaloid,  Brucine,  is  present  in  slightly  smaller  quan- 
tities. In  action  the  two  alkaloids  resemble  each  other  very 
closely  excepting  that  strychnine  is  about  forty  times  as 
strong  as  brucine.  For  this  reason  the  activity  of  nux  vom- 
ica may  be  considered  as  dependent  upon  the  amount  of 
strychnine  present  and  in  the  following  experiments  it  alone 
will  be  considered. 

Examine  the  nux  vomica  bean  and  taste  a  little  of  the 
powdered  drug. 

I.  Place  a  small  amount  of  the  powdered  drug  in  a 
beaker  and  pour  upon  it  10  cc.  of  boiling  water  and  let  it 
stand  until  cold.  Set  this  water  extract  (infusion)  aside 
and  use  it  in  experiments  IV  and  V  given  below.  Place  a 
drop  of  the  infusion  on  the  tongue. 

II.  Prepare  a  fluid  extract  of  nux  vomica  in  the  follow- 
ing manner :  •  . 

Nux  vomica.  No.  60  powder — 50  grams. 

Alcohol  and  water  in  the  proportion  of  2 :  i. 

Moisten  the  powder  with  25  cc.  of  the  diluted  alcohol  and 
let  it  macerate  in  a  well  covered  vessel  in  a  warm  place  for 
48  hours.  Pack  it  in  a  percolator  (see  percolation,  page  44) 
and  gradually  pour  on  diluted  alcohol  until  the  drug  is  ex- 
hausted. Reserve  the  first  35  cc.  of  the  percolate  and  evap- 
orate the  remainder  over  a  water  bath  to  a  soft  extract.  Dis- 


■  EXPERIMENTAL  PHARMA  CO  LOGY.  gy 


98 


LABORATORY  GUIDE  IN 


-  EXPERIMENTAL  PHARMACOLOGY  99 

solve  this  extract  in  the  35  cc.  saved  and  make  the  total  bulk 
up  to  50  cc.  with  diluted  alcohol. 
Define  a  fluid  extract. 

III.  Isolate  the  strychnine  in  the  fluid  extract  in  fol- 
lowing manner : — ■ 

Dilute  the  fluid  extract  with  about  twice  its  own  volume 
of  distilled  water  and  pour  it  into  a  separating  funnel  and 
render  it  alkaline  with  ammonia.  Add  about  25  cc.  of  a 
mixture  of  chloroform  and  ether  in  the  proportion  of  2 :  i 
and  shake  gently  for  three  minutes  and  set  the  funnel  aside 
to  allow  the  chloroformic  solution  to  separate.  When  sep- 
aration has  taken  place,  draw  the  chloroform  and  ether  off 
into  a  beaker  and  repeat  the  agitation  of  the  watery  extract, 
using  a  fresh  supply  of  chloroform  and  ether.  Draw  this 
off  as  before  into  the  breaker  with  the  first  solution.  The 
watery  extract  can  now  be  thrown  away.  Pour  the  chloro- 
form and  ether  into  the  separating  funnel,  which  has  been 
v/ashed,  and  add  about  25  cc.  of  water  and  enough  dilute 
HoSO^  to  make  the  solution  distinctly  acid,  shake  gently 
for  a  few  minutes  and  then  separate  the  water  and  chloro- 
form as  before,  placing  the  acidulated  water  in  a  flask.  Re- 
peat the  agitation  of  the  chloroform  and  ether  with  fresh 
acidulated  water,  adding  the  latter  when  separation  has 
taken  place,  to  the  first  lot  in  the  flask.  (The  waste  chloro- 
form is  to  be  poured  into  a  large  bottle  provided  for  it  and 
later  redistilled.)  As  the  acidulated  watery  solution  is  prob- 
ably colored  it  should  be  shaken  in  the  flask  with  charcoal 
and  filtered.  Render  the  filtrate  distinctly  alkaline  with 
ammonia  and  shake  it  out  twice  as  before  in  the  separating 
funnel,  with  small  quantities  of  the  mixture  of  chloroform 
and  ether.    When  this  has  been  done,  pour  this  chloroformic 


loo  LABORATORY  GUIDE  IN 

solution  into  a  glass  evaporating  dish,  add  to  it  a  drop  of 
concentrated  hydrochloric  acid  and  stir  it  well  with  a  glass 
rod  or  put  the  mixture  in  a  small  flask  and  shake  it  well. 
The  solution  becomes  milky  from  the  formation  of  strych- 
nine hydrochloride,  which  is  thrown  out  of  solution  and 
may  be  separated  from  the  chloroform  by  allowing  the  lat- 
ter to  evaporate  spontaneously  or  by  warming  it  gently  over 
a  water  bath. 

Caution. — In  this  process  two  things  are  of  prime  im- 
portance in  determining  the  success  of  the  manipulations : — 
First,  to  agitate  sufficiently  the  chloroform  and  ammonia  or 
sulphuric  acid  solutions  to  allow  thorough  intermingling  of 
the  solutions.  Three  to  five  minutes  should  be  devoted  to 
each  shaking.  Second,  make  sure  that  the  correct  reaction 
is  present.  Test  with  litmus  paper  each  time  and  after 
shaking  for  a  minute  or  two,  test  again.  (See  Alkaloids, 
Exp.  No.  II.)     The  reaction  must  be  distinct  each  time. 

Explain  why  each  step  in  the  manipulation  was  carried 
out.     (Compare  Alkaloids,  Exp.  Nos.  I,  II,  VII.) 

The  crystallized  strychnine  hydrochloride  is  to  be  used  in 
tests  given  below.     (Exp.  Nos.  X,  XL) 

IV.  To  a  few  cubic  centimeters  of  the  infusion  made  in 
Exp.  I  add  a  few  drops  of  ferric  chloride  solution.  The 
dark  color  of  the  mixture  is  due  to  the  tannic  acid  present 
in  the  nux  vomica  bean  uniting  with  the  iron  to  form  ferric 
tannate.  (To  a  dilute  solution  of  tannic  acid  add  ferric 
chloride.) 

V.  Inject  0.5  cc.  of  the  infusion  into  the  lymph  sac  of  a 
frog,  and  replacing  the  animal  under  a  bell  jar,  observe  the 
effect.    Keep  full  notes.    If  any  convulsions  come  on,  after 


-   EXPERIMENTAL  PHARMACOLOGY.  loi 


J02  LABORATORY  GUIDE  IN 


EXPERIMENTAL  PHARMACOLOGY 


103 


studying  them  carefully,  try  to  find  out  the  point  of  action 
of  the  drug  by  dividing  off  in  turn  the  cerebrum,  optic 
lobes,  and  finally  destroying  the  cord,  watching  the  effect 
of  each  operation  on  the  symptoms.  The  strychnine  con- 
vulsion is  "tonic"  as  distinguished  from  the  "clonic"  type, 
^v-hich  is  caused  by  some  drugs  and  occurs  in  some  diseases. 
(Compare  Camphor (  page  112.) 

Make  a  drawing  of  a  frog  in  a  strychnine  (tonic)  con- 
vulsion. 

VI.  Inject  into  the  lymph  sac  of  a  second  frog  ^  cc.  of 
a  1/10%  solution  of  strychnine  sulphate  diluted  with  physio- 
logical salt  solution  to  make  a  convenient  amount.  In  this 
animal,  as  in  the  previous  experiment,  study  the  early  effects 
of  the  poison  before  convulsions  come  on.  Look  for  changes 
in  irritability,  in  reflexes,  etc.  When  convulsions  come  on, 
paint  the  skin  of  the  animal  with  a  2%  solution  of  cocaine 
observing  any  changes  which  occur.  If  the  convulsions 
cease  wash  the  animal  off  with  water  and  put  it  to  one  side 
observing  it  again  at  the  end  of  10  or  15  minutes.    Explain. 

If  paralysis  should  occur  in  any  of  the  frogs  used  in  these 
experiments  see  if  it  is  due  to  central  or  peripheral  action, 
as  was  done  in  chloral,  Exp.  III. 

VII.  Inject  into  a  small  frog  1/20  mg.  of  strychnine. 
When  convulsions  come  on  place  the  animal  under  a  bell  jar 
with  some  absorbent  cotton  upon  which  has  been  poured 
some  chloroform.  C)bserve  whether  any  change  takes  place 
in  the  convulsions.  If  they  become  lessened,  remove  the 
chloroform  from  the  bell  jar  and  allow  the  animal  to  recover 
from  its  effects.  Do  the  convulsions  return?  When  they  do, 
pith  (page  13)  the  brain  of  the  animal  and  hang  it  up  by  the 
jaw  on  the  hook  for  reflexes.    Now  test  the  animal's  reflexes 


I04 


LABORATORY  GUIDU  IN 


by  pinching  the  toe  and  by  dipping  the  foot  in  acid.  How  do- 
they  differ  from  those  in  a  normal  animal? 

Is  any  therapeutic  hint  contained  in  the  first  part  of  this 
experiment?  In  experiments  Nos.  V,  VI  and  VII,  it  was 
noticed  that  the  same  effects  were  induced  whether  the 
poison  was  employed  as  the  infusion,  or  as  the  pure  alkaloid. 

VIII.^  The  object  of  this  experiment  is  to  apply  strych- 
nine to  the  cervical  portion  of  the  cord  while  leaving  the 
lumbar  cord  unaffected.  Instead  of  pithing  a  frog  as  usual, 
remove  the  brain  entirely  by  cutting  off  the  head  with  a  pair 
of  scissors.  Put  the  lower  blade  in  the  mouth  and  the  upper 
blade  over  the  top  of  the  head,  incline  the  scissors  back- 
ward slightly  and  make  a  quick  cut.  Expose  the  heart  and 
remove  it  from  the  body  in  order  to  stop  the  circulation. 
The  upper  part  of  the  cord  will  be  exposed  by  the  cut  first 
made  and  a  drop  of  a  moderately  strong  strychnine  solu- 
tion (o.i%)  is  to  be  applied  to  it.  After  about  five  minutes 
test  the  reflexes  by  pinching  the  fore  and  hind  legs.  If  the 
experiment  is  successful  a  normal  reflex  should  be  ob- 
tained by  pinching  the  toes  of  the  hind  foot,  while  a  typical 
strychnine  convulsion  involving  the  entire  animal  should 
be  brought  on  by  irritation'  of  the  fore  limbs. 

How  would  such  results  help  to  locate  the  point  of  strych- 
nine activity? 

(See  Pharmacology  and  Therapeutics,  Cushny.  Refer 
to  chapter  on  Strychnine.) 

IX.  With  a  hypodermic  syringe  inject  subcutaneously 
into  a  rabbit  i  mg.  of  strychnine  sulphate.    Watch  the  effect 


^  This  experiment  is  not  always  successful  when  carried  out  by  students  and 
may  be  omitted  if  thought  best.  However,  if  successful,  it  is  of  so  much  in- 
terest and  importance  in  locating  the  point  of  action  of  the  drug  as  to  merit 
a   place   in   these   notes. 


EXPERIMENTAL  PHARMACOLOGY.  105 


jo6  LABORATORY  GUIDE  IN 


-  EXPERIMENTAL  PHARMACOLOGY  107 

on  reflexes,  etc.  If  convulsions  come  on  control  them  with 
a  few  drops  of  chloroform  on  cotton  held  close  to  the  ani- 
mal's nose. 

Compare  the  convulsions  in  this  animal  with  those  seen  in 
the  frog.  Note  the  efficiency  of  chloroform  in  controlling 
the  convulsions  of  strychnine. 

Chemical  Tests  for  Strychnine. 

Xa.  Put  one  or  two  crystals  of  the  strychnine  hydro- 
chloride, prepared  in  Exp.  Ill,  on  a  porcelain  cover  and  add 
to  it  two  drops  of  concentrated  sulphuric  acid,  mixing  the 
two  with  a  clean  dry  glass  stirring  rod.  Put  beside  the 
strychnine-sulphuric  acid  mixture  on  the  cover  a  minute 
crystal  of  potassium  bichromate  and  with  the  glass  rod 
(which  has  been  washed  and  dried)  draw  the  bichromate 
crystal  through  the  strychnine  mixture.  Observe  the  changes 
in  color.  This  "oxidation-reaction"  or  "fading  purple  test" 
for  strychnine  is  most  delicate  and  characteristic.  It  is  said 
to  be  capable  of  detecting  i /20000th  of  a  grain  of  the  alka- 
loid. The  order  of  the  color  changes  is  very  important  in 
making  the  test.  The  purple,  which  should  first  appear, 
changes  to  a  crimson,  which  turns  to  a  cherry  red  that  is 
fairly  persistent. 

Xb.  Carry  out  the  same  experiment  as  above,  using 
manganese  dioxide  instead  of  potassium  bichromate.  Com- 
pare the  results  with  Exp.  Xa. 

This  reaction  is  obtained  with  almost  any  oxidizing  agent, 
such  as  potassium  permanganate,  potassium  f erricyanide,  or 
lead  dioxide.  Manganese  dioxide  is  one  of  the  best  as  the 
changes  in  color  take  place  very  slowly,  in  some  cases  lasting 
some  minutes. 


io8 


LABORATORY  GUIDE  IN 


If  much  brucine  is  present  in  the  preparation,  this  color 
test  is  much  interfered  with  and  it  may  be  impossible  to  get 
the  correct  changes. 

Xc.  After  testing  your  own  alkaloid  with  the  two  re- 
agents named,  make  the  same  tests  on  some  pure  strychnine 
w^hich  will  be  furnished  you.  Compare  the  colors  with 
those  obtained  with  your  preparation. 

XL  Dissolve  the  rest  of  the  strychnine  hydrochloride 
(Exp.  Ill)  in  about  20  cc.  distilled  water  and  put  the  solu- 
tion into  5  test  tubes,  diluting  with  a  little  water  if  neces- 
sary and  test  them  with  the  various  alkaloidal  reagents. 


Reagent 

Precipitate? 

Limit  of  delicacy  of 
reagent 

a 

b 

c 

d- 

e 

Tannic   acid. 

Picric    acid. 

Iodine   in   potassium   iodide. 

Mercury-potassivim    iodide. 

Phosphotungstic   acid. 

1-20,000 
1-100,000 
1-150,000 
1-200,000 

Therapeutic  uses. 

On  account  of  their  intensely  bitter  taste,  preparations  of 
liux  vomica  are  employed  in  cases  of  loss  of  appetite  and 
malnutrition.  By  their  action  on  the  taste  organs  they 
probably  increase  reflexly  the  secretion  of  the  gastric  juice 
and  thus  aid  digestion. 

Strychnine  has  been  largely  used  in  the  past  as  a  stimulant 
to  the  central  nervous  system  in  various  conditions  in  which 
depression  of  the  brain  or  cord  was  present.  It  was  em- 
ployed in  shock  or  collapse,  and  in  failure  of  the  respiratory 
or  of  the  vaso-motor  center.     In  recent  years  considerable 


EXPERIMENTAL  PHARMA  CO  LOGY 


109 


no  LABORATORY  GUIDE  IN 


.  EXPERIMENTAL  PHARMACOLOGY 


III 


doubt  has  been  expressed  as  to  whether  the  drug  exerts 
any  beneficial  action  in  such  conditions  when  administered 
to  man  in  therapeutic  doses.  This  doubt  has  led  to  the 
restriction  of  its  use  but  it  is  still  employed  to  a  certain  ex- 
tent. By  stimulating  the  activity  of  the  spinal  cord  to  a 
slight  extent  it  increases  the  "tone"  of  the  muscles  and  of 
the  blood  vessels  whose  muscular  coats  are  controlled  by 
the  spinal  centers. 


112  LABORATORY  GUIDE  IN 


Camphor. 


Camphor  is  a  white,  translucent,  crystalline  substance  ob- 
tained from  the  volatile  oil  of  the  camphor  tree  (Cin- 
namomum  Camphora).  In  addition  to  its  local  irritant  ac- 
tion, it  exerts  a  very  marked  effect  upon  the  central  nervous 
system  producing  symptoms  which  are  quite  characteristic 
and  which  may  well  be  studied  in  connection  with  those 
produced  in  mammals  by  strychnine. 

I.  Weigh  a  cat  or  rabbit  and  then  administer  to  it 
through  a  stomach  tube  camphor  dissolved  in  olive  oil  (2  G. 
camphor  for  each  kilo  of  body  weight).  Replace  the  ani- 
mal in  a  cage  and  observe  the  early  symptoms  and  the  con- 
vulsions which  should  come  on  in  about  half  an  hour.  Com- 
pare them  carefully  with  those  produced  by  strychnine  and 
try  to  control  them  with  chloroform.  (After  observing 
them  sufficiently  the  animal  should  be  killed  with  the  anaes- 
thetic.) 

A  similar  convulsion  is  also  caused  by  thujon  which  is 
an  isomere  of  camphor  and  the  active  poisonous  principle 
derived  from  the  volatile  oil  of  sage,  tansy,  and  absinth.  It 
may  be  used  in  place  of  the  camphor  in  the  above  experi- 
ment if  desired.  It  is  administered  to  cats  by  means  of  the 
stomach  tube  in  doses  of  about  2  cc.  (Some  preparations  of 
thujon  are  active  in  i  cc.  doses  while  others  require  5  cc.) 
Note  the  marked  change  in  respiration  which  occurs  be- 
fore the  convulsions  come  on. 

It  will  be  seen  that  the  convulsions  caused  by  thujon  are 
also  of  a  distinctly  different  type  from  those  following  a 
poisonous  dose  of  strychnine.  Those  caused  by  the  latter 
drug  are  tonic  in  type  and  are  due  to  an  action  on  the  spinal 


EXPERIMENTAL  PHARMACOLOGY  113 


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EXPERIMENTAL  PHARMACOLOGY 


115 


cord.  On  the  other  hand,  the  thujon  (and  camphor)  con- 
vulsions are  "clonic"  and  are  caused  by  stimulation  of  some 
unknown  part  of  the  cerebrum  or  lower  part  of  the  brain. 

Picrotoxin,  the  active  principle  of  a  climbing  shrub, 
Anamirta  paniculata,  indigenous  to  India,  also  has  a  phar- 
macological action  much  like  the  drugs  mentioned  above, 
and  the  clonic  type  of  convulsion  may  be  very  easily  dem- 
onstrated by  its  use. 

II.  Inject  into  a  frog  i  mg.  picrotoxin  and  replace  the 
animal  under  the  bell  jar.  When  the  convulsions  come  on 
study  them  carefully  indicating  by  a  drawing  the  position 
the  animal  takes.  Remove  in  order  the  different  portions 
of  the  central  nervous  system  by  successive  cuts  as  has  been 
done  in  previous  experiments  and  thus  determine  the  point 
of  action  of  the  drug. 

III.  Inject  subcutaneously  into  a  guinea  pig  5  mg.  of 
picrotoxin.    Study  the  convulsions  as  before. 

Thujon  and  picrotoxin  are  not  employed  in  therapeutics. 
Their  pharmacological  action  would  indicate  that  they  might 
be  of  value  as  stimulants  to  the  central  nervous  system  and 
especially  to  the  respiratory  center.  As  such  they  might  be 
of  value  in  shock  and  collapse  and  perhaps  in  narcotic  pois- 
oning.   Camphor  has  long  been  used  in  shock  and  collapse. 


ii6  LABORATORY  GUIDE  IN 

Opium. 

Opium  is  the  inspissated  juice  obtained  from  the  fruit- 
capsules  of  the  white  poppy  (Papaver  somniferum).  The 
capsules  are  incised  while  yet  unripe  and  the  milky  juice 
allowed  to  exude  and  dry  by  spontaneous  evaporation  until 
it  becomes  a  brownish  gummy  mass. 

The  poppy  is  grown  in  Turkey,  Asia  Minor,  Persia,  India 
and  China,  but  that  obtained  from  Smyrna  and  Turkey  is  by 
far  the  most  valuable.  Persian  and  Indian  opium  is  im- 
ported mainly  as  sources  of  the  opium  alkaloids  and  the 
Chinese  opium  is  consumed  entirely  in  that  country. 

Opium  contains  about  twenty  alkaloids  of  which  Mor- 
phine and  Codeine  are  the  most  important,  and,  indeed,  it 
may  be  said  that  the  value  of  opium  depends  upon  the  per 
cent  of  morphine  which  it  contains.  The  U.  S.  Pharmacopoeia 
specifies  that  the  crude  opium  when  moist  shall  contain  not 
less  than  9.5  per  cent  of  crystallizable  morphine,  while  the 
dried  product  must  assay  from  10  to  10.5  per  cent.  Opium 
from  the  different  localities  varies  exceedingly  in  its  mor- 
phine content. 

Morphine  was  discovered  in  181 7  by  Seitiirner,  of  Eim- 
beck,  Germany,  and  was  named  by  him  morphium.  It  exists 
in  the  opium  principally  in  combination  with  meconic  acid, 
although  some  may  be  present  as  a  lactate  or  a  sulphate,  both 
of  these  acids  being  found  in  the  crude  drug.  Meconic  acid 
is  of  most  importance  as  it  is  strictly  characteristic  of 
opium  and  its  preparations,  and  its  positive  detection  is 
proof  of  the  presence  of  some  opium  preparation. 

In  their  solubilities  morphine  and  codeine  differ  widely 
from  the  other  alkaloids,  for  morphine  is  relatively  insoluble 


.    UXPURIMBNTAI  PHARMACOLOGY  117 


ii8  LABORATORY  GUIDE  IN 


EXPERIMENTAL  PHARMACOLOGY 


119 


in  chloroform  and  even  less  soluble  in  ether  ( 1-6250)  ; 
codeine,  on  the  other  hand,  is  more  soluble  in  water  than 
any  other  alkaloid  except  caffeine. 

Morphine  Reactions. 

I.  Put  a  few  crystals  of  morphine  on  a  porcelain  cover 
and  add  a  drop  or  two  of  pure  concentrated  sulphuric  acid 
and  mix  the  two  with  a  clean  dry  glass  rod.  No  color 
should  be  formed  if  the  morphine  is  absolutely  pure,  but  oc- 
casionally a  faint  pink  tint  is  seen.  Heat  the  mixture  very 
S^ently.  At  about  150°  a  dirty  green  or  rose-red  color  is  de- 
veloped, and  on  still  further  heating,  the  solution  becomes  al- 
most black.  Allow  it  to  cool  and  add  some  water,  when  a 
greenish  blue  color  appears  which  changes  to  green  when 
ammonia  is  added  in  excess. 

II.  Put  a  few  crystals  of  morphine  on  a  cover  and  mix 
them  with  a  drop  or  two  of  concentrated  sulphuric  acid  as  in 
Exp.  I.  To  this  add  a  drop  of  distilled  water  which  will 
heat  the  mixture.  Now  treat  with  a  drop  of  concentrated 
nitric  acid,  which  should  give  a  rose-red  color,  changing  to 
brown. 

(This  reaction  for  morphine  is  very  delicate,  being  well 
shown  by  o.oi  milligram  of  the  alkaloid.) 

III.  To  a  drop  of  concentrated  nitric  acid  on  a  porcelain 
cover  add  a  minute  amount  of  morphine.  An  orange-red 
color  is  obtained,  which  changes  to  yellow  on  heating. 

IV.  (a)  Mix  a  small  amount  of  morphine  with  about 
twice  its  weight  of  cane  sugar.  By  means  of  a  glass  rod  add 
a  drop  of  concentrated  sulphuric  acid.  This  should  produce 
a  purple  color,  changing  gradually  to  blood-red  and  brown- 
ish-red and  becoming  an  olive-brown  on  dilution  with  water. 


I20 


LABORATORY  GUIDE  IN 


(b)  The  above  test  can  be  modified  as  follows : — To  a 
solution  of  morphine  add  cane  sugar  until  the  solution  is 
saturated.  Now  pour  this  solution  carefully  down  the  side 
of  a  second  test  tube  in  the  bottom  of  which  is  concentrated 
sulphuric  acid.  By  inclining  the  second  tube  the  solution 
can  be  poured  in  so  that  no  mixture  of  the  two  fluids  takes 
place  and  a  purple  or  rose-red  ring  will  form  at  their  junc- 
tion.   This  ring  becomes  more  intense  on  standing. 

V.  Dissolve  some  morphine  sulphate  in  a  little  water  and 
add  it  to  a  solution  of  iodic  acid.  What  color  does  it  turn? 
Add  to  the  resulting  mixture  some  thin  starch  paste.  What 
was  the  first  color  due  to? 

Note. — In  order  to  color  the  starch  paste  blue  morphine  must  be  present  in 
the  strength  of  i  to  looo.  If  no  blue  is  obtained  mix  the  solution  well  and 
pour  down  the  side  of  the  test  tube  some  very  dilute  ammonia.  A  double  ring 
will  be  seen  at  the  junction  of  the  two  fluids;  it  will  be  blue  below  and  brown 
above. 

VI.  To  a  few  cubic  centimeters  of  water  add  some  mor- 
phine sulphate  and  to  this  solution  add  a  few  drops  of  ferric 
chloride  solution.    A  deep  blue  color  is  obtained. 

VII.  Meconic  acid  reaction.  To  a  cubic  centimeter  of 
tincture  of  opium  diluted  with  a  little  water  add  two  drops 
of  ferric  chloride  solution.  What  color  does  the  solution 
become?  If  the  color  is  obscured  by  a  precipitate,  filter 
tiie  solution. 

VIII.  Inject  into  the  anterior  lymph  sac  of  a  frog  15  mg 
of  morphine  sulphate.  Replace  the  animal  under  the  bell 
jar  and  examine  it  from  time  to  time,  comparing  the  ef- 
fects with  those  caused  by  chloral  or  strychnine,  and  with 
those  seen  in  the  frog  injected  with  codeine  in  Exp.  IX. 

Note. — This  experiment  (VIII)  should  be  begun  early  in  the  laboratory  ses- 
sion as  it  sometimes  takes  two  or  three  hours  for  the  full  effects  to  appear. 


.  nXPURIMBNTAL  PHARMACOLOGY    .  121 


122  LABORATORY  GUIDE  IN 


,  EXPERIMENTAL  PHARMACOLOGY 


123 


IX.  Inject  in  the  anterior  lymph  sac  of  a  frog  i  cc.  of 
a  1.5%  solution  of  codeine  phosphate  and  replace  the  animal 
under  the  bell  jar.  Compare  the  symptoms  in  this  animal 
with  those  seen  in  the  morphine  frog  noting  especially  the 
relative  time  of  onset  of  the  symptoms  in  the  two. 

Among  the  alkaloids  found  in  opium  which  are  of  less 
importance  than  those  studied  is  thebaine,  which  resembles 
strychnine  very  closely  in  its  pharmacological  action.  In 
fact,  many  of  the  alkaloids  of  opium  may  be  arranged  in  a 
series  with  the  depressant,  morphine,  at  one  end  of  the  series 
and  thebaine  at  the  opposite  end.  A  comparison  of  the  ef- 
fects of  thebaine  with  those  produced  by  morphine  and 
codeine  may  be  made  by  injecting  i  cc.  of  a  1.5%  solution 
of  thebaine  into  a  frog  and  comparing  it  with  the  animals 
studied  in  Exp.  VIII  and  IX. 

X.  Inject  subcutaneously  into  a  dog  50  mg.  of  morphine 
sulphate  and  observe  the  effects  keeping  an  accurate  protocol 
of  the  symptoms. 

XIa.  Inject  30  to  40  mg.  of  morphine  sulphate  sub- 
cutaneously into  a  cat.  Put  the  animal  in  a  wire  cage  and 
compare  the  effects  with  those  seen  in  the  dog.  (Note  un- 
der Exp.  VIII  appHes  here  also.) 

Xlb.  Instead  of  injecting  a  cat  a  dose  of  from  5  to  10 
mg.  of  morphine  sulphate  may  be  given  a  white  mouse. 
Symptoms  much  like  those  produced  in  the  cat  are  seen,  the 
main  difference  being  that  they  appear  in  from  5  to  10  min- 
utes. 

The  peculiar  action  of  morphine  on  the  cat  is  not  confined 
to  that  animal,  but  is  found  in  all  the  members  of  the  cat 
tribe,  and  also  in  the  horse  and  ass. 


124  LABORATORY  GUIDE  IN 

Examine  a.  crude  opium, 

b.  powdered  opium, 

c.  extract  of  opium, 

d.  tincture  of  opium  (Laudanum), 

e.  camphorated  tincture  of  opium  (Paregoric), 

f.  powdered  opium  and  ipecac  (Dover's  pow- 

der), 
♦  g.  codeine, 

h.  and  herome. 

Note  the  odor  of  all  the  opium  preparations  and  taste  d, 
e,  and  f. 

Therapeutics. 

The  symptoms  induced  by  opium  in  man  resemble  more 
closely  those  in  the  dog  than  those  in  the  other  animals.  It 
depresses  the  brain,  and  for  this  reason  is  used  in  cases  of 
sleeplessness  when  this  condition  results  from  pain,  because 
in  depressing  the  cerebrum  it  acts  on  the  pain  centers,  for 
which  it  seems  to  have  a  special  affinity.  In  the  dog  the 
respiration  was  slowed  in  the  later  stages  of  the  action  from 
depression  of  the  respiratory  center,  and  opium  prepara- 
tions are  often  employed  in  cough  mixtures  to  allay  exces- 
sive irritability  of  this  part  of  the  medulla.  The  numerous 
other  purposes  for  which  opium  and  morphine  are  used 
cannot  be  illustrated  experimentally. 


nXPBRIMBNTAL  PHARMACOLOGY  125 


126  LABORATORY  GUIDE  IN 


.  EXPERIMENTAL  PH'ARMACOLOGY  127 

Curara. 

Curara,  the  South  American  arrow  poison,  varies  in  com- 
position with  the  different  locaHties  from  which  it  is  ob- 
tained. The  best  known  product  comes  from  Guiana  and  is 
prepared  from  the  bark  of  Strychnos  toxifera,  a  tree  native 
to  that  country.  Curara  as  obtained  in  commerce  consists  of 
dark  brown,  shining,  brittle  masses  which  contain  vegetable 
extracts  in  addition  to  two  alkaloids,  tubocurarine  and 
curine.  The  characteristic  action  of  the  drug  is  dependent 
principally  upon  the  first  alkaloid  mentioned,  the  latter  hav- 
ing quite  a  different  action  and  being  much  weaker. 

Not  only  do  solutions  of  curara^  deteriorate  on  keeping, 
but,  what  is  of  greater  importance,  the  crude  drug,  as  ob- 
tained in  the  market,  varies  greatly  in  strength,  many  of  the 
preparations  being  entirely  inert.  There  is  no  chemical 
method  by  which  the  activity  of  curara  can  be  estimated ; 
ihe  only  test  is  pharmacological.  Each  solution  to  be  used 
has  to  be  "standardized"  by  tests  on  animals  and  the  dose 
necessary  to  produce  characteristic  effects  ascertained. 

I.  Inject  into  a  frog  a  poisonous  dose  of  a  standardized 
solution  of  curara.  Study  the  eff"ect  on  spontaneous  move- 
ments, reflexes,  ability  to  turn  over  when  placed  on  its  back, 
and  length  of  time  leg  muscles  will  remain  contracted  when 
stimulated  by  applying  the  tetanizing  current  to  the  lumbar 
spine.  If  paralysis  should  occur  see  if  it  is  due  to  central  or 
peripheral  action  (see  Soporifics,  Exp.  Ill,  page  92).  This 
animal  is  to  be  replaced  under  the  bell  jar  and  kept  moist,  as 
it  will  probably  recover  in  a  few  days. 


^  The    curara    solution    should    be    made    up    in    physiological    salt    solution    to 
which  some  thymol   has  been  added. 


128  LABORATORY  GUIDE  IN 

II.  Pith  a  frog,  tie  it  on  the  frog  board  and  expose  the 
sciatic  nerve  and  artery  along  the  back  of  the  thigh  by  a 
short  longitudinal  incision  through  the  skin,  and  careful 
separation  of  the  muscles  with  the  dissecting  needle.  Sepa- 
rate the  sciatic  nerve  from  the  surrounding  tissues  along  a 
short  distance  of  its  course  and  then  pass  a  thread  under 
the  nerve  and  tie  it  tightly  around  the  rest  of  the  thigh  as 
high  up  as  possible. 

Free  the  animal,  inject  curara  as  in  I,  and  from  time  to 
time  compare  the  reflexes  in  the  hind  legs.  Stimulate  the 
lumbar  cord  with  the  electric  current  and  note  any  differ- 
ences between  the  two  sides.  See  whether  the  reflexes  re- 
main equal  in  the  two  legs  and  whether  cross  reflexes  can  be 
obtained. 

If  the  experiment  is  successful  the  operated  leg  should  not 
be  affected  by  the  poison  and  should  respond  to  electrical 
stimulation.  In  such  a  case  cut  off  the  leg  just  above  the 
knee,  leaving  attached  to  it  the  sciatic  nerve  which  has  been 
carefully  separated  from  the  thigh  muscles  and  cut  toward 
the  upper  part  of  its  course.  Holding  the  leg  preparation 
by  the  severed  end  of  the  femur  lay  the  hanging  sciatic 
nerve  for  a  few  moments  in  the  curara  solution.  Remove  it 
and  stimulate  it  with  the  electric  current.  Does  curara  affect 
the  nerve  trunk?  Dip  the  muscle  in  the  solution  and  after 
a  few  moments  stimulate  the  sciatic  nerve  again.  Is  there 
any  change?  If  so,  test  the  activity  of  the  muscles  by  direct 
stimulation.    Where  does  curara  act? 

Examine  the  heart  of  the  animal  in  Exp.  II. 

Examine  Curara. 


EXPERIMENTAL  PHARMACOLOGY.  129 


130  LABORATORY  GUIDE  IN 


.  EXPERIMENTAL  PHARMACOLOGY  131 

Therapeutics. 

The  main  importance  of  curara  is  in  physiological  experi- 
ments. It  has  been  suggested,  and  in  some  few  cases  used, 
to  allay  the  convulsions  due  to  tetanus  or  to  strychnine 
poisoning.  It  is  not  recommended  for  such  conditions  as 
its  action  is  too  uncertain  and  too  dangerous. 


132  LABORATORY  GUIDE  IN 

Nicotine. 

The  alkaloid,  nicotine,  is  obtained  from  the  dried  leaf  of 
the  tobacco  plant  (Nicotianum  Tabacum)  in  which  it  is 
found  to  the  extent  of  from  2  to  8  per  cent.  The  pure  alka- 
loid is  a  colorless,  oily  fluid,  which,  on  exposure  to  air,  be- 
comes yellowish  or  brown.  It  has  a  strong,  unpleasant  odor 
resembling  somewhat  that  of  tobacco. 

In  an  aqueous  solution  the  alkaloid  gives  a  strong  alkaline 
reaction  and  it  unites  with  various  acids  to  form  salts. 

I.  Inject  0.5  cc.  of  nicotine  chloride  solution^  into  the 
anterior  lymph  sac  of  a  frog.  Observe  the  effects,  noting 
especially  the  posture  of  the  animal  in  the  early  stages  of 
poisoning  as  this  is  very  characteristic  of  nicotine  action. 
In  addition,  watch  for  any  twitching  of  the  muscles,  and  if 
paralysis  should  come  on  see  whether  it  is  due  to  central  or 
peripheral  action. 

Make  a  drawing  of  the  animal  in  the  early  stage  of  the 
nicotine  action  when  the  legs  are  drawn  up  over  its  back. 

The  action  of  nicotine  upon  the  sympathetic  nervous  sys- 
tem is  studied  later  (pages  248,  260). 

Therapeutics. 
Nicotine  is  not  employed  in  therapeutics. 


^  The  nicotine  chloride  solution  is  formed  by  neutralizing  a  small  quantity  of 
nicotine  with  1-20  normal  hydrochloric  acid.  Each  cubic  centimeter  will  then 
contain  about  4   mgs.    of   nicotine. 


EXPERIMENTAL  PHARMACOLOGY. 


133 


134  LABORATORY  GUIDE  IN 


-    BXPBRIMBNTAl  PHARMACOLOGY  135 

Veratrine. 

Vcratrine  is  an  alkaloid  prepared  from  the  seeds  of  Asa- 
groea  officinalis.  It  is  a  grayish  powder  which  causes  very 
marked  irritation  when  even  a  minute  quantity  is  inhaled. 

The  alkaloid  is  not  of  great  medical  importance  but  is  of 
pharmacological  interest,  owing  to  a  peculiar  action  it  pos- 
sesses which  can  be  best  studied  in  cold-blood  animals,  al- 
though the  same  effect  may  be  elicited  in  mammals. 

I.  Inject  I  mg.  of  veratrine  sulphate  (i  cc.  of  0.1%  sol.) 
into  a  frog.  Watch  and  describe  the  effects.  Observe  the 
awkwardness  of  its  movements  which  appears  in  a  few  min- 
utes. When  the  clumsiness  is  well  developed  locate  the 
point  at  which  the  drug  acts  by  removing  the  cerebrum,  optic 
lobes,  and  finally  destroying  the  spinal  cord  if  the  symptoms 
do  not  disappear  earlier.  Stimulate  the  sciatic  plexus  with 
single  shocks  from  the  induction  coil,  comparing  the  results 
with  those  seen  in  a  normal  frog.  Repeat  the  stimulation 
of  the  sciatic  nerve  several  times  at  short  intervals  study- 
ing the  effect  of  fatigue  upon  the  veratrinized  muscle. 
Finally,  after  allowing  the  muscle  to  rest,  stimulate  the 
muscle  directly,  using  single  shocks.  At  what  point  does 
the  drug  act? 

Therapeutics. 

The  phenomena  seen  in  this  experiment  give  no  hint  as  to 
the  use  of  the  drug  in  therapeutics ;  it  is  employed  for  ex- 
ternal application,  and  internally  to  a  limited  extent  for  its 
effects  on  the  circulation. 


136 


LABORATORY  GUIDE  IN 


Caffeine. 

Caffeine  differs  from  the  other  alkaloids  in  the  fact  that  it 
is  contained  in  a  number  of  plants  belonging  to  different 
species.  It  is  obtained  from  the  berry  of  the  coffee  (Coffea 
Arabica)  ;  from  the  leaves  of  tea  (Thea  Chinensis),  besides 
being  present  in  the  kola  nut  of  Central  Africa;  in  the 
Paraguay  tea  and  Guarana  paste  of  South  America  and  in 
the  Apalache  tea  of  Virginia  and  Carolina. 

In  some  of  these  plants  it  is  associated  with  two  closely 
related  alkaloids,  TJieobromine  and  Theophyllin,  which  re- 
semble caffeine  very  much  in  some  of  their  actions.  All 
three  alkaloids  are  derivatives  of  xanthine,  which  in  turn 
is  closely  related  to  uric  acid. 

Caffeine  is  a  very  feeble  base  and  forms  salts  only  with 
difficulty  and  these  salts,  as  a  rule,  are  very  unstable,  being 
easily  decomposed,  even  by  water. 

I.     Test  the  solubility  of  caffeine  m 


Reagent. 


Solubility. 


Water,    cold. 
"        hot. 
Alcohol. 
Chloroform. 


II.  Put  100  mg.  caffeine  in  a  test  tube  and  add  4  cc. 
cold  water  to  it.  Shake.  Now  add  100  mg.  sodium  benzoate 
and  shake.  What  therapeutic  use  could  be  made  of  this 
reaction  ? 

III.  Compare  in  like  manner  the  solubility  of  theo- 
bromine with  that  of  the  double  salt,  theobromine  sodium 
salicylate,  and  note  the  advantage  the  latter  might  possess. 


.     EXPERIMENTAL  PHARMACOLOGY.  137 


138 


LABORATORY  GUIDE  IN 


.   EXPERIMENTAL  PHARMACOLOGY 


139 


IV.     Apply  the  various  alkaloidal  reagents  to  a  1%  solu- 
tion of  caffeine. 


Reagent. 

Precipitation? 

a 

Tannic   acid. 

b 

Picric  ncid. 

c 

Iodine  in  potassium  iodide. 

d 

Mercury-potassium  iodide. 

e 

Phosphotungstic   acid. 

Note. — In  its  reaction  to  c  and  d  caffeine  differs  from  all  other  alkaloids 
except   theobromine   and   colchicine. 

V.  Murexoin  test.  To  a  few  crystals  of  caffeine  on  the 
porcelain  cover  add  a  drop  of  concentrated  nitric  acid.  Hold 
the  cover  with  a  pair  of  forceps  some  distance  above  the 
flame  of  a  Bunsen  burner  and  thus  gently  evaporate  the 
mixture  to  dryness.  After  cooling  the  porcelain  cover  al- 
low the  residue  to  come  in  contact  with  strong  ammonia 
fumes  and  note  the  color  obtained. 

This  reaction  is  given  by  the  members  of  the  xanthine 
series  and  is  practically  identical,  with  that  given  by  uric 
acid. 

VI.  Inject  5  nig.  of  caffeine  (i  cc.  0.5%  soi.)  into  a 
frog.  Study  and  describe  the  symptoms  comparing  them 
with  those  induced  by  strychnine  and  veratrine.  See  whether 
the  peculiar  change  in  the  muscles  is  due  to  an  action  on 
the  nervous  system  by  destroying  its  various  parts  as  was 
done  with  veratrine.  Excise  a  small  piece  of  the  affected 
muscle  and  tease  it  in  salt  solution  on  a  microscopic  slide. 
Examine  it  under  the  microscope,  using  the  low  power  lens, 
and  compare  its  appearance  with  normal  muscle  prepared 
in  the  same  way.  (For  an  explanation  of  the  changes  con- 
sult Pharmacology  and  Therapeutics,  Chapter  on  Caffeine.) 


I40  LABORATORY  GUIDE  IN 

The  action  of  caffeine  and  its  allies  on  the  heart  and  011 
the  kidney  will  be  studied  later  (pages  207,  240). 
Examine  the  Coffee  bean  (Coffea  Arabica)  ; 

Tea  leaves  (Thea  Chinensis)  ; 

Caffeine  citrate; 

Theobromine. 

Theobromine  sodium  salicylate. 

Therapeutic  Uses. 

Caffeine  is  largely  employed  as  a  central  nervous  stimu- 
lant. The  stimulant  action  on  the  highest  parts  of  the  brain 
is  more  noticeable  in  man  than  in  the  frog  as  the  cerebrum 
ill  the  latter  is  not  as  highly  differentiated  and  the  principal 
symptoms  are  due  to  cord  action.  In  man,  however,  caffeine 
(usually  taken  in  the  form  of  coffee  or  tea)  stimulates  the 
cerebrum  and  the  medulla  and,  to  a  less  extent,  the  spinal 
cord.  It  is  of  great  use  in  cerebral  depression,  such  as  is 
found  in  shock  and  collapse.  In  narcotic  poisoning,  also,  its 
action  on  the  medullary  centers  is  especially  valuable.  Both 
caffeine  and  theobromine  are  largely  used  for  their  stimu- 
lant action  on  the  kidneys  by  which  they  increase  the  urine. 
They  are,  therefore,  of  service  in  removing  abnormal  col- 
lections of  fluid  from  the  body,  such  as  oedema  or  ascites, 
whether  of  cardiac  or  renal  origin. 


EXPERIMENTAL  PHARMACOLOGY.  141 


142  LABORATORY  GUIDE  IN 


,  nXPBRIMBNTAL  PHARMACOLOGY  143 

Cocaine. 

Cocaine  is  the  principal  alkaloid  derived  from  the  leaves 
of  Erythroxylon  coca,  a  shrub  which  is  a  native  of  Peru  and 
Bolivia  and  of  other  parts  of  South  America.  It  is  very 
unstable,  being  quite  prone  to  undergo  chemical  change,  so 
tiiat  preparations  of  the  drug  frequently  contain  the  products 
of  its  decomposition.  For  this  reason  there  are  no  very 
delicate  or  distinctive  chemical  tests  by  which  the  drug  may 
be  identified.  Being  an  alkaloid,  it  is  thrown  out  of  solution 
by  the  various  precipitants  of  the  group,  and  in  its  behavior 
to  solvents  it  resembles  in  general  the  other  alkaloids. 

Cocaine  has  an  important  local  action  upon  the  termina- 
tions of  sensory  nerves,  which  makes  it  of  very  great  use  in 
medicine. 

I.  Take  a  piece  of  filter  paper  2  or  3  cm.  square  and 
moisten  it  with  a  few  drops  of  a  4%  cocaine  hydrochloride 
solution  and  place  it  upon  the  tip  of  the  tongue.  Retain  it 
in  this  position  for  two  or  three  minutes  and  then  examine 
the  condition  of  that  part  of  the  tongue  exposed  to  the  drug, 
testing  it  especially  as  to  the  sense  of  touch,  temperature,  and 
taste.  Contrast  its  effect  on  the  taste  of  sugar  and  of  nux 
vomica. 

II.  Place  a  cat  in  a  cat  box  allowing  the  head  to  pro- 
trude. After  examining  the  corneal  reflex  and  the  reaction 
of  the  iris  to  bright  and  dim  light,  put  into  one  eye  2  drops 
of  a  4%  solution  of  cocaine  hydrochloride,  keeping  the 
other  eye  as  a  control.  After  5  minutes  examine  the  eye 
testing  the  reflexes  to  touch  and  to  both  dim  and  bright  light 
and  comparing  the  size  of  the  pupil  with  the  control.     If 


144 


LABORATORY  GUIDE  IN 


the  drug  effects  are  not  sufficiently  well  marked  make  a 
second  or  even  third  application  of  cocaine  allowing  5  or 
ID  minute  intervals  to  elapse  to  allow  of  absorption.  What 
therapeutic  uses  may  be  made  of  these  actions  of  cocaine? 

In  addition  to  these  local  actions  cocaine  has  also  a  marked 
effect  on  the  central  nervous  system  which  may  be  illustrated 
by  the  following  experiment: 

III.  Inject  subcutaneously  into  a  white  mouse^  from 
3  to  5  mg.  of  cocaine.  Place  the  animal  in  a  suitable  con- 
tainer and  observe. 

Therapeutic  Uses. 

Cocaine  is  moat  largely  employed  in  medicine  for  its  action 
in  paralyzing  the  sensory  nerve  endings  causing  a  local 
anaesthesia,  permitting  of  operations  being  performed  with- 
out pain.  It  is  also  employed  to  dilate  the  iris  in  order  that 
examinations  of  the  interior  of  the  eye  may  be  made.  It 
is  rarely  used  for  its  action  on  the  nervous  system. 


^  Somewhat  similar  symptoms  may  be  obtained  in  the  dog  by  the  injection 
of  cocaine  in  doses  of  from  20  to  25  mg.  per  kg.  If  convulsions  come  on 
they  may  be   controlled   with   chloroform. 


.    EXPBRIMBNTAL  PHARMACOLOGY 


145 


j^g  LABORATORY  GUIDE  IN 


.    nXPBRIMBNTAL  PHARMACOLOGY  147 

The  Belladonna  Series. 

Several  plants  belonging  to  the  Solanacese  furnish  alka- 
loids which  are  very  closely  related,  not  only  chemically,  but 
also  in  their  pharmacological  effects.  Chief  among  these 
alkaloids  is  Atropine  which  is  contained  in  the  leaves  and 
roots  of  the  deadly  nightshade  (Atropa  Belladonna).  Next 
in  importance  to  atropine  are  Hyoscyamine  and  Hyoscine. 
These  alkaloids  are  all  obtained  from  other  sources  than 
that  named,  viz.;  henbane  (Hyoscyamus  niger),  the  thorn- 
apple  (Datura  Stramonium)  and  other  plants  of  less  im- 
portance. They  are  very  hard  to  isolate  in  absolutely  pure 
form  because  hyoscyamine  is  very  prone  to  change  to  atro- 
pine during  the  manipulations. 

As  stated  above,  they  resemble  one  another  closely  in  theii 
]")roperties  and  actions,  so  that  atropine  alone  will  be  studied. 
Examine  Belladonna  leaves  and  roots. 

I.  Prepare  the  alcoholic  extract  of  belladonna  leaves  ac- 
cording to  the  U.  S.  P.,  using  50  G.  of  the  powdered  drug. 

Define  an  "extract"  of  a  drug. 

II.  Take  some  of  the  extract,  an  amount  about  the  size 
of  a  pea  and  rub  it  up  in  5  cc.  of  salt  solution  and  put  a  drop 
of  the  watery  extract  in  a  cat's  eye  and  watch  for  any 
changes  comparing  it  with  the  normal  eye.  The  cat  is  best 
put  in  a  cat  box  (footnote,  page  12)  with  the  head  exposed 
during  the  experiment;  drop  the  solution  near  the  outer 
canthus  of  the  eye  so  that  it  will  have  to  cross  the  eyeball 
before  escaping  by  the  nasal  duct. 

This  action  of  the  atropine  series  upon  the  pupil  is  known 
as  mydriasis  and  the  drugs  producing  it  are  mydriatics.     It 


148  LABORATORY  GUIDE  IN 

is  the  most  delicate  test  known  for  the  group,  although  a 
few  other  drugs,  of  which  cocaine  is  the  most  important,  act 
as  mydriatics,  though  to  a  less  marked  degree. 

III.  Isolate  the  atropine  from  the  extract  of  belladonna 
by  the  "shaking-out"  process  as  described  below.  Observe 
the  precautions  mentioned  under  the  isolation  of  strychnine 
(page  lOo). 

Mix  the  extract  thoroughly  with  about  lOO  cc.  of  distilled 
water  and  render  it  alkaline  with  ammonia.  Put  the  mix- 
ture in  a  separating  funnel  and  add  20  cc.  of  chloroform. 
Shake  gently  for  3  to  5  minutes  and  set  the  funnel  aside  to 
allow  the  chloroform  to  separate,  drawing  it  off  into  a  flask. 
Add  15  cc.  more  chloroform  to  the  watery  extract  and  shake 
as  before,  and  draw  off  the  chloroform  into  the  flask  with 
the  first  lot.     The  watery  extract  can  now  be  thrown  away. 

Put  the  chloroform  containing  the  alkaloid  in  the  sepa- 
rating funnel  and  add  about  50  cc.  of  water  rendered  dis- 
tinctly acid  with  sulphuric  acid.  Agitate  the  mixture  several 
minutes  as  before,  drawing  off  the  chloroform  from  the 
water  when  separation  has  taken  place.  Repeat  the  process 
with  the  chloroform  and  50  cc.  of  fresh  acidulated  water. 
(The  waste  chloroform  is  to  be  placed  in  a  jar  on  the  side 
table  for  redistillation.)  If  the  watery  solution  is  colored, 
even  slightly,  shake  it  in  a  flask  with  powdered  charcoal  and 
filter  it.  The  filtrate  containing  the  atropine  is  now  ren- 
dered alkaline  with  ammonia  and  shaken  out  again  with  20 
cc.  of  chloroform,  which  is  drawn  off  into  a  glass  evaporat- 
ing dish.  Repeat  the  process  with  10  cc.  of  chloroform  and 
add  it  to  the  20  cc.  in  the  dish  and  set  the  whole  amount 
aside,  protected  from  the  dust,  to  allow  the  chloroform  to 
evaporate  and  the  atropine  to  crystallize. 


EXPERIMENTAL  PHARMACOLOGY  149 


I50  LABORATORY  GUIDB  IN 


.  nXPBRIMBNTAL  PHARMACOLOGY  151 

IV.  Place  a  few  crystals  of  atropine  (from  Exp.  Ill) 
on  a  porcelain  cover  and  add  to  them  a  drop  of  concentrated 
nitric  acid  and  evaporate  it  to  dryness  over  the  water  bath. 
Allow  the  cover  to  cool  and  then  touch  the  residue  with  a 
glass  rod  which  has  been  dipped  in  an  alcoholic  solution  of 
caustic  potash.  A  rich  violet  color  is  produced,  changing  to 
a  dark  red,  which  finally  fades  away,  but  can  be  reproduced 
by  the  addition  of  fresh  alcoholic  potash.  This  reaction 
(known  as  Vitali's  test)  is  almost  peculiar  to  the  atropine 
series  and  is  said  to  be  given  by  o.oooi  mg.  of  the  alkaloid. 

V.  Inject  into  a  frog  15  mg.  of  atropine  sulphate  and  de- 
scribe all  the  symptoms  which  are  observed  following  the  in- 
jection of  the  drug.  Keep  the  animal  for  several  days  until 
complete  recovery  takes  place.  Compare  the  effects  with 
those  induced  by  other  drugs  used  earlier  in  the  course. 

VI.  Count  the  heart  and  respiratory  rates  in  a  dog 
which  is  resting  quietly  and  then  inject  hypodermically  i 
mg.  atropine  sulphate  per  Kg.  body  weight.  After  the 
drug  has  had  time  to  be  absorbed  repeat  the  observations 
previously  made  and  if  necessary  take  further  counts  later 
keeping  a  record  of  the  findings.    Explain  the  results. 

VII.  Take  o.ooi  G.  of  atropine  sulphate  (which  will  be 
furnished  you)  and  describe  all  the  symptoms  experienced. 
Count  the  rate  of  your  heart  before  and  after  taking  the 
drug  and  see  if  there  is  any  change. 

VIII.  Dissolve  the  remainder  of  the  atropine  isolated 
in  about  20  cc.  of  distilled  water  which  has  been  rendered 
acid  with  sulphuric  or  hydrochloric  acid.  Divide  the  solu- 
tion into  five  parts,  put  it  in  test  tubes  and  add  the  alkaloidal 
precipitants  as  follows : 


152 


LABORATORY  GUIDE  IN 


Eeagent. 

Precipitate? 

a 

Tannic    acid. 

b 

Picric    acid. 

c 

Iodine  in  potassium  iodide. 

d 

Mercury-potassium  iodide. 

e 

Phosphotungstic    acid. 

The  action  of  atropine  on  the  innervation  of  the  heart  is 
studied  on  the  turtle  (page  196)  ;  and  on  the  frog  (page 
187). 

Its  action  on  secretions  will  be  studied  on  the  salivary 
gland  (page  263). 

Its  mydriatic  effect  when  applied  locally  was  shown  in 
Exp.  II,  and  the  same  effect  from  internal  administration 
is  studied  under  the  heading  "Cervical  Sympathetic  Nerves," 
(page  251). 

Chemically  atropine  consists  of  a  combination  of  tropine 
with  tropic  acid.    Other  alkaloids  have  been  made  artificially 
by  the  substitution  of  other  acids  in  the  place  of  tropic  acid. 
Ihe  most  important  of  these  synthetic  alkaloids  is  Homa- 
tropine  in  which  tropine  is  combined  with  mandelic  acid. 
,In  many  of  its  actions  it  resembles  atropine  quite  closely, 
the  main  point  of  difference  being  that  its  effects  are  much 
more  transient.     Its  main  use  is  in  ophthalmology  where  it 
is  employed  to  dilate  the  iris  and  paralyze  the  accommoda- 
tion by  acting  on  the  ciliary  body. 
Examine  Atropa  Belladonna, 
Hyoscyamus  niger. 
Datura  Stramonium, 

the  alcoholic  extract  of  belladonna  leaves, 
tincture  of  belladonna  leaves  and 
the  extract  and  tincture  of  hyoscyamus. 
Homatropine  hydrobromide. 


£XPBRIMBNTAL  PHARMACOLOGY  153 


154  LABORATORY  GUIDE  IN 


MXPBRIMBNTAL  PHARMACOLOGY  155 

Therapeutic  Uses. 

Owing  to  the  fact  that  atropine  has  such  a  wide  range  ot 
activities,  its  uses  in  medicine  are  very  numerous.  Among 
these  a  few  may  be  mentioned  as  being  indicated  by  the  va- 
rious experiments  in  which  the  drug  is  employed.  It  is  used 
as  a  stimulant  in  the  depressed  state  of  the  central  nervous 
system  occurring  in  shock,  collapse  and  in  narcotic  poison- 
ing. (Compare  strychnine  and  caffeine.)  For  its  peri- 
pheral action  it  is  employed  for  many  purposes.  In  cases  of 
extreme  slowness  of  the  heart  (Bradycardia)  due  to  over- 
activity of  the  vagus,  atropine  may  be  employed.  It  is  used 
to  dilate  the  pupil  of  the  eye  to  allow  of  ophthalmoscopic 
examination,  or  as  a  therapeutic  agent  in  the  treatment  of 
many  eye  diseases. 

In  excessive  secretion  of  the  saliva  such  as  occurs  in 
poisoning  with  mercury,  and  in  the  night  sweats  of  tuber- 
culosis, atropine  is  very  valuable. 

Finally  it  is  used  in  cases  of  poisoning  by  pilocarpine  or 
muscarine,  the  latter  being  an  alkaloid  resembling  pilocar- 
pine in  many  of  its  actions.  ' 


156  LABORATORY  GUIDE  IN 

Cinchona. 

In  the  plants  belonging  to  the  cinchona  family  are  a  num- 
ber of  species  the  bark  of  which  yields  some  twenty-five 
alkaloids.  Quinine  is  by  far  the  most  important  of  these. 
The  Cinchonacas  are  natives  of  South  America  and,  as  far 
as  is  known,  were  introduced  into  medicine  about  1630. 

Quinine  may  be  regarded  as  a  typical  alkaloid  and  its  be- 
havior towards  various  solvents  and  precipitants  has  already 
been  examined  (page  20). 

In  addition  to  those  properties,  which  it  shares  in  common 
with  the  other  alkaloids,  it  shows  a  few  reactions  of  its  own 
which  are  more  or  less  characteristic. 

Taste  quinine  and  quinine  hydrochloride. 

I.  Weigh  out  0.1  G.  quinine,  the  same  amount  of  quinine 
sulphate,  and  of  quinine  hydrochloride  and  put  them  sepa- 
rately in  three  test  tuDes.  Add  10  cc.  of  water  to  each  test 
tube  and  shake.     Which  is  the  most  soluble? 

II.  To  0.1  G.  quinine  sulphate  in  a  test  tube,  add  about 
10  cc.  distilled  water  and  about  i  cc.  of  dilute  sulphuric  acid. 
Note  the  change  in  solubility  compared  with  I  and  describe 
the  solution  formed.  Solutions  showing  this  peculiar  fluor- 
escence are  also  formed  by  a  few  of  the  other  cinchona 
alkaloids. 

III.  Make  a  solution  of  quinine  sulphate  in  a  test  tube, 
adding  a  drop  or  two  of  sulphuric  acid  if  necessary.  Add 
very  carefully  potassium  hydroxide  to  make  the  solution  as 
nearly  neutral  as  possible  (not  alkaline!).  Add  a  few  drops 
of  chlorine  water  and  after  shaking  add  ammonia  to  excess. 


nXPBRIMUNTAL  PHARMACOLOGY  157 


158 


LABORATORY  GUIDE  IN 


.BXPBRIMBNTAL  PHARMACOLOGY  159 

Observe  the  greenish  coloration  of  the  solution  or  the  green 
precipitate  which  will  be  formed  if  enough  quinine  is  present. 
This  reaction  is  of  importance,  as  it  can  be  employed  in 
testing  for  quinine  in  the  urine  in  cases  in  which  it  is  im- 
portant to  know  that  the  drug  is  being  absorbed. 
Examine  Cinchona  bark. 

Tincture  of  cinchona. 

Compound  tincture  of  cinchona. 

Quinine  tannate   (Tasteless  quinine). 

Euquinine    (Quinine  ethyl  carbonate). 

Therapeutic  Uses. 

The  tinctures  of  cinchona  are  largely  employed  in  medi- 
cine on  account  of  their  bitter  taste ;  they  are  given  espe- 
cially in  cases  of  lack  of  appetite.  The  other  therapeutic 
uses  of  cinchona  and  its  alkaloids  cannot  be  demonstrated 
very  satisfactorily  in  the  laboratory. 

The  tannate  of  quinine  and  euquinine  may  be  used  in 
case  tasteless  preparations  are  desirable  as  in  children. 


i6o  LABORATORY  GUIDB  IN 

Anaesthetics. 

The  most  important  members  of  the  group  of  drugs  used 
to  produce  general  anaesthesia  are  Chloroform  and  Ether. 
Their  depressant  action  on  the  central  nervous  system  will 
be  observed  in  some  of  the  experiments  in  which  they  will 
be  used  to  supplement  the  other  drugs  used  as  anaesthetics 
in  animals  (chloretone,  etc.).  The  cardiac  effects  are 
studied  in  the  frog,  turtle  and  dog.  In  these  animals  it 
will  be  noticed  that  the  heart  dilates  and  becomes  very  weak 
before  there  is  very  much  change  in  the  rate.  The  im- 
portance of  this  in  practical  anaesthesia  is  that  information 
as  to  the  condition  of  the  heart  cannot  be  derived  entirely 
from  the  pulse  rate,  as  the  heart  may  be  seriously  affected 
and  yet  show  very  little  change  in  the  number  of  its  con- 
tractions per  minute.  The  quality  of  the  pulse  must  also 
be  noted. 

The  fact  that  chloroform  acts  very  much  more  strongly 
on  the  heart  than  ether,  will  also  be  demonstrated  in  the 
experiments  mentioned  above. 

Some  of  the  early  eff'ects  of  the  anaesthetics  are  shown  in 
the  following  experiment : — 

I.  Place  a  rabbit  on  the  table  and  hold  it  by  placing  your 
hand  under  its  chest  in  such  a  way  that  you  can  count  its 
heart  and  respiration.  Pour  some  chloroform  on  a  little 
absorbent  cotton  and  hold  it  to  the  animal's  nose  for  a  few 
moments  and  observe  any  change  in  the  rhythm  of  the  heart 
or  respiration. 

Repeat  the  experiment  using  ether  and  afterwards  am- 
monia. It  will  be  seen  that  the  same  effeccs  are  produced  by 
all  three  drugs.    They  are  caused  by  a  reflex  stimulation  of 


.  EXPERIMENTAL  PHARMACOLOGY.  i6i 


j52  laboratory  guide  in 


EXPBRIMBNTAL  PHARMACOLOGY  163 

the  vagus  and  respiratory  centers  from  the  action  of  the  irri- 
tant fumes  on  the  mucous  membranes : 

Other  drugs  that  are  used  as  general  anaesthetics  are 
Nitrous  oxide  gas  and  Bthyl  chloride. 

Nitrous  Oxide. 

The  action  of  nitrous  oxide  may  be  observed  by  placing 
a  frog,  white  mouse,  or  small  guinea  pig  in  a  wide  mouth 
bottle  which  is  fitted  with  a  cork  through  which  pass  two 
pieces  of  glass  tubing.  One  of  these  tubes  is  connected 
with  a  tank  of  nitrous  oxide  gas.  When  the  animal  has 
been  placed  in  the  bottle  and  the  cork  adjusted,  nitrous  oxide 
gas  may  be  admitted  by  opening  the  valve  very  gradually. 
The  outlet  glass  tube  should  be  left  open. 

Observe  the  animal  closely  as  the  percentage  of  gas  in- 
creases in  the  jar,  noting  especially  changes  in  the  rate  of 
respiration.  Keep  a  record  of  the  time  it  takes  to  produce 
complete  ansesthesia  and  when  that  stage  is  reached  turn  off 
the  gas  and  remove  the  animal  from  the  jar.  Note  the 
time  it  takes  for  the  animal  to  recover. 

This  experiment  may  be  varied  by  connecting  a  tank  of 
oxygen  to  the  inlet  tube  in  addition  to  the  nitrous  oxide 
and  admitting  small  percentages  of  oxygen  to  the  bottle  at 
the  same  time  as  the  nitrous  oxide  is  passing  in.  Note  the 
difference  in  symptoms  from  those  seen  when  nitrous  oxide 
alone  was  given. 

Bthyl  Chloride. 

The  action  of  ethyl  chloride  in  producing  general  anses- 
thesia may  be  demonstrated  as  follows : — 

Place  a  white  mouse  in  a  small  beaker  which  has  a  little 
absorbent  cotton  in  the  bottom  of  it,  and  then  spray  a  few 


i64  LABORATORY  GUIDE  IN 

drops  of  ethyl  chloride  (Kelene)  upon  the  cotton.  Note 
the  symptoms  shown  by  the  animal  and  the  length  of  time 
it  takes  to  produce  full  anaesthesia.  Immediately  this  stage 
is  reached  remove  the  animal  and  observe  the  time  it  takes 
to  recover. 

Magnesium  Sulphate  Anesthesia. 

Depression  of  the  central  nervous  system  and  general 
anaesthesia  may  be  produced  in  rabbits  by  the  subcutaneous 
or  intramuscular  injection  of  magnesium  sulphate  in  doses 
of  1.7  G.  per  kilo  of  body  weight.  This  dose  can  be  con- 
siderably lessened  if  sodium  oxalate  is  administered  at  the 
same  time  as  the  magnesium  is  given.  The  anaesthesia  and 
depression  may  be  entirely  recovered  from  if  a  calcium  salt 
be  given  later  as  calcium  antagonizes  the  magnesium  action. 

Experiment : — 

Inject  intramuscularly  into  a  rabbit  1.2  G.  magnesium 
sulphate  per  Kg.  body  weight  and  0.2  G.  sodium  oxalate 
per  Kg.  In  from  i^  to  2  hours  the  animal  should  be  in  a 
state  of  complete  anaesthesia.  Now  inject  slowly  either 
into  an  ear  vein  or  into  a  jugular  6  or  8  cc.  of  a  3%  solu- 
tion of  calcium  chloride.  Note  the  immediate  effect  upon 
the  respiration  and  upon  the  general  condition  of  the  animal. 

Local  Anesthesia. 

Ethyl  chloride  may  also  be  used  to  produce  local  anaes- 
thesia. This  action  is  explained  by  the  great  volatility  of  the 
substance  so  that  when  it  is  sprayed  upon  the  skin  it  evap- 
orates very  rapidly  causing  extreme  chilling  and  finally 
freezing  of  the  part. 

Holding  a  tube  of  ethyl  chloride  in  one  hand  direct  a 
fine  spray  of  the  drug  against  the  back  of  the  other  hand. 


EXPERIMENTAL  PHARMACOLOGY.  165 


l56  LABORATORY  GUIDE  IN 


nXPBRIMBNTAL  PHARMACOLOGY  167 

The  tube  will  have  to  be  held  at  a  distance  of  18  inches  or 
2  feet  from  the  object  sprayed.  In  a  very  few  moments 
the  area  of  skin  will  become  white  and  examination  of  its 
sensitiveness  by  means  of  a  pin  will  show  complete  anaes- 
thesia as  compared  with  the  surrounding  normal  area. 

The  action  of  other  local  anaesthetics  was  illustrated  with 
cocain.  The  action  of  this  drug  and  of  its  alhes,  novocaine, 
etc.,  is  entirely  different  from  ethyl  chloride  as  it  is  due  to 
a  paralysis  of  the  nerve  endings  conveying  the  sensations 
of  pain  and  touch. 


i68  LABORATORY  GUIDE  IN 

The  Digitalis  Series. 

This  series  embraces  a  large  number  of  drugs  which  re- 
semble each  other  in  their  pharmacological  action.  While 
they  act  on  various  organs  in  the  body,  the  cardiac  action  is 
characteristic  of  the  group  and  distinguishes  it  from  all 
others.  The  members  of  the  series  are  derived  from  plants 
widely  distributed  in  nature  and  as  widely  separated  in  their 
botanical  relationships. 

The  most  important  is  digitalis,  which  is  obtained  from 
Digitalis  purpurea  (purple  fox  glove)  ;  strophanthus  from 
Strophanthus  hispidus ;  and  squills  from  Scilla  maritima. 

I.  Prepare  the  tincture  of  digitalis  according  to  the  U.  S. 
P.,  using  25  G.  of  the  powdered  drug  and  the  proportionate 
amount  of  75%  alcohol.  (Made  by  adding  one  volume  of 
water  to  three  of  alcohol.) 

Define  a  tincture. 

This  tincture  is  to  be  used  in  the  following  experiments : 

II.  To  5  cc.  of  the  tincture  add  15  cc.  of  distilled  water, 
is  a  precipitate  formed?     Cork  and  set  aside  the  mixture 

'and  examine  it  after  a  few  days. 

III.  Examine  the  tincture  for  the  presence  of  tannic 
acid  by  adding  ferric  chloride. 

IV.  To  5  cc.  of  the  tincture  add  two  drops  of  mercury- 
potassium  iodide.  Is  a  precipitate  formed?  What  does  the 
result  indicate? 

V.  Evaporate  over  a  water  bath  10  cc.  of  the  tincture  to 
about  one-half  its  volume  and  then  make  up  the  concen- 
tiated  tincture  to  the  original  bulk  by  adding  physiological 
salt  solution. 


EXPERIMENTAL  PHARMACOLOGY.  169 


j-O  LABORATORY  GUIDE  IN 


-  EXPERIMENTAL  PHARMACOLOGY  171 

Now,  pith  a  frog  and  tie  it  on  a  board.  Expose  the  heart 
and  inject  into  a  lymph  sac  i  cc.  of  the  modified  tincture. 
Record  all  changes  in  the  heart. 

Describe  any  change  in  the  rate  of  the  organ  and  also  any 
differences  in  the  size  of  the  various  chambers  or  in  their 
manner  of  contracting  or  dilating. 

VI.     Biological  assay  of  tincture  of  digitalis. 

Digitalis  preparations  vary  considerably  in  their  strength 
and  it  is  impossible  to  standardize  them  by  any  chemical 
means  such  as  can  be  employed  in  the  cases  of  those  drugs 
whose  activity  depends  upon  alkaloids  which  may  be  isolated 
in  pure  form  and  weighed. 

It  is  very  hard  to  isolate  the  glucosides  in  pure  form  as 
they  are  likely  to  break  up,  making  the  results  obtained  very 
unreliable.  For  this  reason  a  pharmacological  or  biological 
estimation  of  the  strength  of  a  preparation  is  employed  as 
follows : — • 

Evaporate  10  cc.^  of  the  tincture  over  a  water  bath  to 
about  half  volume-  and  then  make  it  up  to  the  original  vol- 
ume by  the  addition  of  physiological  salt  solution.  Select 
3  frogs  of  about  the  same  size,  weigh  them  carefully  (within 
0.5  G.)  and  copy  the  markings  on  the  back  of  each  animal 
so  as  to  be  able  to  identify  them  later.  Into  the  anterior 
lymph  sacs  of  the  frogs  inject  by  means  of  a  glass  pipette 
doses  of  the  modified  tincture  as  follows : — ■ 

The  doses  to  be  given  have  to  be  calculated  according  to 
the  weight  of  the  frogs  used.     Frog  No.   i   is  to  receive 


^  All    measurements    must    be    made    with    the    same    attention    to     accuracy 
as  in  quantitative  chemical  analyses. 
="  Why? 


172  LABORATORY  GUIDE  IN 

0.004  cc/  multiplied  by  the  number  of  grams  body  weight 
of  the  animal;  No.  2  receives  0.0055  cc.  per  gram  body 
weight,  and  No.  3  0.007  cc.  per  gram  body  weight.  (These 
small  quantities  may  be  most  accurately  measured  by  dilut- 
ing the  modified  tincture  still  more  with  salt  solution  and 
then  injecting  the  proportionately  larger  dose.  The  amount 
of  fluid  given  each  animal  should  measure  about  0.5  cc.) 

Keep  a  jrecord  of  the  time  when  each  injection  is  made. 
Replace  the  frogs  under  the  bell  jar  and  at  the  end  of  one 
hour  from  the  time  of  each  injection  examine  the  condition 
of  each  frog's  heart.  Pith  the  animal  if  necessary,  tie  it  on 
the  frog  board  and  expose  the  heart  according  to  the  usual 
method.  To  have  a  correct  "end  reaction''  the  heart  should 
have  just  ceased  beating  with  the  ventricle  in  systole  and  the 
two  auricles  markedly  distended  with  blood.  Light  me- 
chanical stimulation  of  the  ventricle  may  call  out  a  local 
contraction  but  there  should  be  no  general  beat  of  the 
ventricle.  (Compare  Exp.  V.)  If  all  three  hearts  are  still 
beating  when  they  are  examined  the  doses  were  too  small 
and  must  be  increased  in  fresh  frogs  of  corresponding 
.weight.  If,  on  the  other  hand,  the  frogs  have  been  dead 
for  some  time,  the  doses  were  too  large  and  must  be  made 
smaller  in  other  frogs. 

Having  ascertained  now  by  this  method  the  toxic  strength 
of  a  tincture  it  may  be  adjusted  by  dilution  or  concentration 
so  that  its  strength  may  correspond  to  any  standard  which 
may  have  been  adopted. 

The  method  of  assay  described  is  of  considerable  im- 
portance as  it  or  some  of  its  modifications  is  employed  by 


^  Three  section  of  the  class  may  assay  tne  same  tincture  by  varying  the 
doses  to  be  administered  by  0.0005  cc.  so  as  to  fill  in  the  gaps  in  the  figures 
given   above.      In   this   way   a   complete   assay   may   be   obtained. 


EXPERIMENTAL  PHARMACOLOGY.  173 


jy^  LABORATORY  GUIDE  IN 


.EXPERIMENTAL  PHARMACOLOGY  ij^ 

pharmaceutical  firms  for  the  standardization  of  the  cardiac 
remedies  of  the  group  (see  page  272). 

The  remainder  of  the  tincture  is  to  be  saved  to  be  used  in 
subsequent  experiments  on  blood  pressure,  etc. 

The  effects  of  digitalis  on  the  mammalian  heart,  both 
when  normal  and  also  when  in  a  state  of  auricular  fibrilla- 
tion and  on  the  arterial  blood  pressurv^  are  studied  later. 
(Exps.,  pages  207  and  200.)  Its  effect  on  the  excretion  of 
urine  (diuretic  action)  may  be  studied  in  the  rabbit  (note, 
page  239),  and  the  vascular  changes  in  the  intestinal  and 
renal  vessels  according  to  directions  given  on  pages  299  and 
304- 


176  LABORATORY  GUIDB  IN 

Infusion  of  Digitalis. 

Another  preparation  of  digitalis  which  is  largely  used  in 
some  parts  of  the  world  in  place  of  the  tincture  and  which 
it  resembles  in  most  of  its  actions  is  the  Infusion. 

I.  Prepare  the  infusion  of  digitalis  acording  to  the  U. 
S.  P. 

Define  an  infusion. 

II.  Test  the  infusion  for  glucosides  with  tannic  acid. 

III.  Fill  a  test  tube  about  half  full  with  the  infusion, 
place  the  thumb  over  the  mouth  of  the  tube  and  shake  it  up 
thoroughly.  Note  the  soap-like  character  of  the  foam.  The 
infusion  contains  a  glucoside,  digitsaponin,  which  possesses 
the  property  of  forming  a  frothy  solution  and  of  holding  in- 
soluble bodies  in  suspension. 

IV.  Pith  a  frog,  tie  it  on  the  board  and  expose  the  heart 
as  usual.  Inject  into  a  lymph  sac  0.5  cc.  of  the  infusion. 
Compare  the  efifects  with  those  noticed  in  the  experiments 
in  which  the  tincture  was  employed. 

V.  The  infusion  of  digitalis  may  be  assayed  by  the  meth- 
od described  under  "Tincture"  (Exp.  VII,  page  171),  em- 
ploying, of  course,  correspondingly  larger  doses  of  the  in- 
fusion. Also,  in  making  the  assay,  it  will  not  be  necessary 
to  evaporate  the  preparation  as  was  done  with  the  tincture. 
Why? 

VI.  Save  the  remainder  of  the  infusion  and  examine  it 
from  time  to  time  and  note  any  changes  taking  place  in  it. 

Examine  digitalis  leaves  (Digitalis  purpuras)  ; 

strophanthus  seeds   ( Strophanthus  hispidus)  ; 
and  squills,  the  sliced  bulb   (Scilla  maritima). 


EXPERIMENTAL  PHARMACOLOGY  177 


178  LABORATORY  GUIDE  IN 


.  EXPERIMENTAL  PHARRIACOLOGY  179 

Also  examine  the  following  preparations : — 
Tincture  of  strophanthus ; 
Tincture  of  squills; 
Syrup  of  squills. 

Therapeutic  Uses. 

Digitalis  and  its  allies  are  employed  principally  in  certain 
affections  of  the  heart.  When  a  valve  of  the  heart  is  diseased 
so  that  it  does  not  perform  its  functions  properly,  the  heart 
has  an  increased  amount  of  work  to  do.  In  most  cases  its 
walls  increase  in  thickness  (hypertrophy)  to  allow  of  the 
extra  work  being  done,  in  other  words  the  heart  becomes 
"compensated."  But  if  an  unusual  demand  is  made  on  the 
heart  before  it  has  time  to  hypertrophy,  the  walls  stretch  on 
account  of  their  weakness,  and  the  heart  is  said  to  become 
"dilated."  When  this  occurs  the  contractions  are  weak  and 
imperfect  and  the  result  is  that  the  circulation  becomes 
slowed  and  the  blood  tends  to  collect  on  the  venous  side  of 
the  vascular  system,  resulting  in  a  passive  congestion  of  the 
organs,  and  in  marked  cases  a  generalized  oedema  of  the 
body  may  follow. 

In  such  conditions  digitalis  is  of  the  greatest  value.  As 
was  seen  in  its  effect  on  the  frog's  heart,  it  increases  the 
strength  of  the  contractions  and  in  some  cases  lessens  the 
extent  of  dilatation.  The  heart  begins  to  pump  more  blood 
into  the  arteries,  thus  improving  the  nutrition  of  the  body 
cells.  One  of  the  first  organs  to  be  benefited  is  the  heart 
itself  through  its  coronary  arteries.  From  its  improved 
blood  supply  it  becomes  stronger  and  may  be  able  to 
hypertrophy  and  thus  carry  on  its  increased  work  without 
the  further  use  of  digitalis.     The  improved  circulation  re- 


i8o  LABORATORY  GUIDE  IN 

suits  in  a  disappearance  of  the  congestion  of  the  organs  and 
of  the  oedema. 

The  removal  of  the  oedema  is  also  aided  by  the  diuretic 
action  of  the  drugs  belonging  to  this  series ;  this  appears  to 
be  due  mainly  to  the  cardiac  action  (cardiac  diuretic). 

The  use  of  the  drug  in  conditions  of  auricular  fibrillation 
is  demonstrated  in  a  later  experiment. 


EXPERIMBNTAL  PHARMACOLOGY  i8i 


i82  LABORATORY  GUIDB  IN 


.  EXPERIMENTAL  PHARMACOLOGY  183 

Aconite. 

Several  alkaloids  are  derived  from  plants  belonging  to  the 
Aconitum  genus,  and  among  them  Aconitine  is  the  most  im- 
portant. It  is  obtained  from  the  root  of  the  monk's  hood 
(Aconitmii  Napellus). 

The  most  important  preparation  containing  aconitine  is 
the  tincture  of  aconite,  the  alkaloids  being  very  rarely  used 
as  different  preparations  vary  considerably  in  activity,  owing 
to  the  fact  that  aconitine  decomposes  with  great  readiness. 

I.  To  2  cc.  of  the  tincture  of  aconite  add  an  equal  amount 
of  water.  Try  to  dissolve  the  precipitate  formed  by  adding 
some  95%  alcohol.  The  precipitate  in  this  case  consists  of 
resinous  bodies  (extractives)  which  are  thrown  out  of  so- 
lution by  the  water  but  which  are  redissolved  by  the  alcohol. 

II.  Add  10  drops  of  tincture  of  aconite  to  about  i  cc.  of 
distilled  water  in  a  clean  beaker.  Take  the  solution  in  the 
mouth  and  retain  it  there  withovit  swallowing  for  about  a 
ri]inute,  after  which  it  may  be  ejected  and  the  mouth  rinsed 
with  water.  Observe  whether  any  unusual  sensation  comes 
on  in  from  five  to  ten  minutes  in  that  part  of  the  mouth 
with  which  the  aconite  solution  has  come  in  contact. 

These  symptoms,  which  are  due  to  stimulation  of  the  sen- 
sory nerve  endings,  are  characteristic  of  two  drugs,  aconite 
and  veratrine. 

III.  Pith  a  frog,  tie  it  on  the  board  and  expose  the  heart 
by  the  usual  method  (page  14).  Inject  into  a  lymph  sac 
0.5  cc.  of  tincture  of  aconite  and  keep  a  record  of  all  changes 
taking  place  in  the  organ,  noting  not  alone  changes  in  rate, 
but  also  in  the  character  of  the  contractions. 


184  LABORATORY  GUIDE  IN 

After  some  minutes  (10)  the  heart  may  become  very  ir- 
regular, this  irregularity  showing  especially  in  the  ventricle. 
If  this  change  in  the  rhythm  does  not  develop  in  a  reason- 
able time  it  may  be  necessary  to  give  a  second  injection  of 
the  aconite.  The  observation  of  this  phenomenon  should 
not  be  omitted.    Keep  the  heart  moist  with  salt  solution. 

IV.     Biological  assay  of  aconite. 

The  class  may  carry  out  a  biological  assay  of  the  tincture 
of  aconite  according  to  the  method  described  in  the  Phar- 
macopceia.  Three  guinea  pigs  may  be  injected  after  they 
have  been  carefully  weighed.  The  dose  given  the  first  shall 
be  the  standard  dose_  demanded  in  the  Pharmacopoeia,  viz. : 
0.0004  cc.  per  gram  body  weight  of  the  animal ;  for  the  sec- 
ond 0.0002  cc.  per  gram ;  and  for  the  third  0.0006  cc.  per 
gram.  These  doses  will  give  a  rough  estimate  of  the  strength 
of  the  tincture,  and  this  estimate  may  be  still  further  nar- 
rowed down  by  subsequent  injections  into  fresh  animals 
employing  doses  determined  by  the  results  obtained  in  the 
first  series  of  animals. 

Examine  the  root  of  Aconitum  Napellus. 

Therapeutics. 

Aconite  is  occasionally  used  in  cases  of  neuralgia  on  ac- 
count of  its  action  on  sensory  nerve  endings. 

It  has  been  used  more  or  less  extensively  in  the  past  to 
slow  the  heart  and  to  lower  the  temperature  in  cases  of 
fever.  It  is  doubtful  whether  it  accomplished  either  of 
the  effects  when  given  in  therapeutic  doses  and  its  use  has 
therefore  been  largely  given  up  in  favor  of  the  newer  and 
stronger  drugs  of  the  antipyrine  group. 


.  UXPBRIMBNTAL  PHARMACOLOGY  1 85 


1 86  LABORATORY  GUIDE  IN 


nXPBRIMBNTAL  PHARMACOLOGY  1S7 

The  Effect  of  Drugs  on  the  Frog's  Heart. 

Pith  a  frog,  tie  it  on  a  board  and  expose  the  heart  as 
usual.  Dissect  out  the  vagus  nerve  and  stimulate  it  with  a 
weak  tetanizing  current  from  the  induction  coil.  To  find  the 
vagus  nerve  in  the  frog:  expose  the  heart,  split  the  pectoral 
girdle  entirely  and  draw  the  anterior  limbs  well  apart.  Care- 
fully remove  the  skin  and  connective  tissue  in  the  neck  and 
two  large  nerves  (9"  and  12'')  will  be  seen  on  either  side 
passing  forward.  Trace  these  backward  and  they  will  be 
found  to  take  a  turn  outward  to  enter  the  base  of  the  skull. 
The  vagus  in  this  position  lies  between  them,  running  par- 
allel to  them  in  this  part  of  their  course.  The  electrical 
stimulation  of  the  nerve  should  be  with  a  weak  current  and 
not  unduly  prolonged  as  the  nerve  is  fatigued  very  easily. 

Describe  the  changes  in  the  heart  caused  by  vagus  stimu- 
lation. 

Note. — In  a  certain  number  of  cases  the  vagus  in  the  frog  is  not  active 
and  it  will  therefore  be  impossible  to  affect  the  heart  by  its  electrical  stimula- 
tion. See  that  the  heart  is  kept  moist  with  salt  solution  and  that  it  is  not 
injured   by    instruments    or    rough    handling   as    it   is   very    easily    damaged. 

In  this  frog  or  in  a  second  one  if  necessary,  examine  the 
action  of  chloroform  by  putting  the  animal,  with  the  heart 
exposed,  under  the  bell  jar  in  which  there  is  a  piece  of  ab- 
sorbent cotton  soaked  in  chloroform. 

Record  all  changes  seen  in  the  heart,  whether  changes  in 
rate  or  in  the  condition  of  the  various  chambers. 

If  the  effects  become  very  marked,  remove  the  animal 
from  the  bell  jar  and  allow  the  heart  to  recover.  Keep  it 
moist  by  dropping  on  it  a  small  amount  of  physiological  salt 
solution. 

When  the  organ  has  recovered  repeat  the  experiment,  us- 
ing ether  instead  of  chloroform. 


1 88  LABORATORY  GUIDE  IN 

Compare  the  actions  of  the  two  drugs.  (See  Anaesthet- 
ics, page  i6o).  Also  compare  the  action  of  chloral  on  the 
heart.     (Page  92.) 

After  the  heart  has  returned  once  more  to  normal,  put  a 
drop  of  a  pilocarpine  (or  muscarine)  solution  on  it  and  note 
any  change  in  rate.  After  a  few  minutes  add  a  few  more 
drops  of  the  same  solution  and  note  any  further  change. 
Compare  the  appearance  of  the  heart  with  its  condition  un- 
der vagus  stimulation.  If  the  heart  should  stop  contracting, 
put  two  drops  of  an  atropine  solution  on  the  organ  and  if 
necessary,  after  a  few  minutes  make  a  second  or  third  ap^ 
plication  of  atropine. 

If  the  heart  begins  to  beat  once  more  apply  pilocarpine 
solution  again  and  see  if  it  will  stop  it.  Also  try  stimulation 
of  the  vagus  nerve.  If  pilocarpine  will  not  stop  the  heart, 
try  chloroform  by  exposing  the  organ  to  the  fumes  as  be- 
fore. 

From  your  knowledge  of  the  anatomy  and  physiology  of 
the  vagus  nerve  try  to  account  for  the  effects  induced  by 
the  drugs  as  seen  above,  considering  them  in  the  following 
order,  pilocarpine,  atropine,  pilocarpine  and  chloroform. 

The  effects  of  digitalis,  aconite,  chloral  and  paraldehyde 
on  the  heart  are  studied  elsewhere.     (Pages  171,  183,  92.) 


nXPBRIMBNTAL  PHARMACOLOGY  189 


igo  LABORATORY  GUIDB  IN 


.  BXPBRIMBNTAL  PHARMACOLOGY  191 

The  Effect  of  Drugs  on  the  Turtle's  Heart. 

Destroy  the  brain  of  a  turtle  by  a  blow  with  a  hammer, 
and  turning  the  animal  on  its  back,  tie  it  on  an  operating 
board.  Draw  the  head  out  so  as  to  put  the  neck  on  the 
stretch  and  fasten  it  in  this  position  by  means  of  a  nail 
driven  through  it.  Remove  the  lower  shell  (plastron),  cut- 
ting the  lateral  attachments  with  bone  forceps  or  a  saw; 
after  cutting  the  hard  parts  the  shell  can  be  raised  slightly 
and  the  underlying  soft  parts  severed  with  a  scalpel,  taking 
care  to  cut  toward  the  shell.  Carefully  cut  away  the  skin 
and  fascia  near  the  base  of  the  neck,  and  if  the  coracoids 
(G)  and  clavicles  (F)  are  in  the  way  they  may  be  cut  back 
with  bone  forceps  and  a  scalpel.  Large  white  muscles 
(B.  B.),  the  long  retractors  of  the  head  (Retrahens  capitis 
collique)  will  now  be  exposed,  running«back  from  the  head 
and  placed  deeply  in  the  neck  on  either  side  of  the  median 
line.  Two  other  large  white  muscles  (coraco-hyoideus.  A) 
will  also  be  seen  passing  on  either  side  from  the  front  legs 
forward  toward  the  median  line  of  the  neck.  Two  nerves 
(C,  D)  may  be  found  emerging  from  under  the  long  re- 
tractors just  posterior  to  the  point  where  the  coraco-hyoidei 
unite  in  the  median  line.  In  the  upper  part  of  their  course 
they  lie  internal  to  the  long  retractors,  then  wind  around 
the  muscle  to  reach  its  upper  surface  (in  this  position  of  the 
animal)  and  pass  down  the  neck,  lying  on  the  muscle.  One 
of  these  nerves  is  the  vagus  (D),  which  must  be  identified 
by  gently  separating  it  from  the  surrounding  structures  and 
stimulating  it  with  the  electric  current,  watching  the  efifect 
on  the  heart.  When  it  is  found,  place  a  ligature  around  it. 
In  dissecting  out  the  nerve  be  careful  not  to  take  hold  of  it 


192 


LABORATORY  GUIDE  IN 


Vvith  forceps  as  this  injures  it.  If  it  is  necessary  the  fascia 
in  the  vicinity  of  the  nerve  may  be  grasped  with  the  forceps 
and  then  torn  away  from  the  nerve  by  means  of  the  dissect- 
ing needle. 

Expose  the  heart  by  cutting  away  the  pericardium.  Thread 
a  small  curved  needle  with  about  six  or  eight  inches  of 


Fig.  No.  6.     Turtle  with  plastron  removed  and  vagi  dissected  out.     A.  Coraco- 

hyoideus   muscle,    B.    Retrahens   capitis    collique    muscle    (right   muscle    is 

cut   across),   C.    Sympathetic    nerve,    D.    Vagus,   E.    Carotid    artery, 

F.    Clavicle,    cut    end,    G.    Coracoid,    cut    end,    H.    Heart. 


EXPERIMENTAL  PHARMACOLOGY.  193 


^^^  LABORATORY  GUIDB  IN 


EXPERIMENTAL  PHARMACOLOGY 


195 


medium  thread  and  take  a  stitch  in  either  side  of  the 
ventricle,  trying  to  include  between  them  the  area  of  great- 
est contraction.  Tie  the  thread  in  the  heart  with  a  loose 
knot,  leaving  the  two  free  ends  which  are  to  be  attached  to 
the  myocardiograph  (Fig.  7). 


Fig.  No.  7.     Myocardiograph  for  turtle's  heart. 


Place  the  board  with  the  turtle  on  the  stand  of  the  myo- 
cardiograph and  arrange  the  instrument  so  that  the  ends  of 
the  two  levers  (A  and  B)  shall  be  beside  the  ventricle.  Tie 
the  threads  attached  to  the  ventricle  to  the  ends  of  the  two 
levers.  In  making  this  attachment  see  that  the  heart  is  tied 
up  close  to  the  levers  and  that  considerable  space  is  not 
allowed  between  because  the  threads  are  not  drawn  tightly. 
The  writing  lever  now  begins  to  move  downwards  with 
each  contraction  and  upwards  with  each  relaxation  of  the 


196  LABORATORY  GUIDE  IN' 

ventricle.  Vary  its  height  so  that  it  will  record  the  move- 
ments of  the  heart  on  the  smoked  surface  of  a  drum  properly 
placed.  Arrange  the  stationary  lever  (D)  to  draw  a  base 
line  just  below  the  tracing  of  the  cardiac  movements. 
-  After  taking  a  short  tracing  showing  the  normal  con- 
tractions of  the  heart,  take  one  showing  the  effects  of  stimu- 
lation of  the  vagus. 

Note. — Mark   on  the   tracing  when   any   stimulation   is   begun   or  stopped   or 
when   any    drug    is   applied. 

After  the  heart  has  recovered  and  while  the  record  is 
continuing  apply  slowly  8  or  lo  drops  of  a  0.5%  nicotine 
solution  near  the  base  of  the  heart  where  the  large  vessels 
enter.  This  should  be  sufficient  to  cause  distinct  slowing 
of  the  heart. 

After  the  heart  has  recovered  from  the  nicotine  try  the 
effect  of  vagus  stimulation  again. 

Continuing  the  tracings,  apply  a  few  drops  of  a  pilocar- 
pine solution  (0.5%)  to  the  heart.  When  the  contractions 
become  very  infrequent  (and  not  until  then)  or  stop  entirely, 
apply  atropine  (0.1%)  to  the  heart,  adding  a  drop  or  two  at 
a  time  to  the  organ. 

When  the  contractions  return  to  normal,  stimulate  the 
vagus  with  the  electric  current  as  before. 

Apply  pilocarpine  again  to  the  heart.  Compare  the  trac- 
ings with  those  obtained  earlier  in  the  experiment. 

Shake  i  cc.  of  chloroform  in  20  or  30  cc.  of  physio- 
logical salt  solution  and  pour  some  of  this  solution  from 
time  to  time  over  the  heart.  When  the  cardiac  contractions 
become  very  weak,  remove  the  chloroform  solution  with  a 
piece  of  absorbent  cotton  and  assist  the  recovery  of  the  heart 
by  bathing  it  in  fresh  salt  solution. 


EXPERIMENTAL  PHARMACOLOGY. 


197 


198 


LABORATORY  GUIDE  IN 


-  EXPERIMENTAL  PHARMACOLOGY  199 

Note  that  pilocarpine  does  not  stop  the  heart  after  atro- 
pine, nor  has  vagus  stimulation  any  effect.  Chloroform,  on 
the  other  hand,  acts  after  atropine.  How  is  this  to  be  ex- 
plained?   How  did  nicotine  produce  its  effect? 

Remove  carefully  the  paper  with  the  tracings  from  the 
drum  and  fix  it  by  passing  it  through  a  solution  of  shellac 
in  alcohol. 

Study  the  tracings  according  to  directions  given  on 
page  215. 


200  LABORATORY  GUIDE  IN 

The  Effect  of  Drugs  on  Blood  Pressure. 

Anzesthetize  a  dog,  cat  or  rabbit  according  to  the  methods 
described  earlier  (page  ii),  and  when  the  animal  is  com- 
pletely under  the  influence  of  the  anaesthetic,  tie  it  on  its 
back  on  the  operating  board  and  remove  the  hair  from  the 
neck  region.  Make  an  incision  about  four  inches  long  in 
the  median  line  of  the  neck  and  expose  the  trachea ;  insert 
a  tube  (page  iS)  to  allow  of  artificial  respiration  should  it 
become  necessary. 

Expose  the  jugular  vein  on  one  side  and  insert  a  cannula 
(page  i6).  On  the  opposite  side  expose  the  carotid  artery 
and  in  like  manner  tie  a  cannula  in  it  (page  17). 

Fill  the  venous  cannula  with  physiological  salt  solution 
and  the  carotid  cannula  with  a  solution  made  by  mixing 
equal  volumes  of  water  and  a  saturated  solution  of  sodium 
sulphate.     (See  note  2,  page  ry.) 

The  ordinary  form  of  mercury  manometer,  which  is  so 
well  known  as  to  need  no  description,  is  used.  Fill  the 
proximal  limb  of  the  U-shaped  tube,  i.  e.,  the  limb  which  is 
to  be  connected  with  the  blood  vessel,  with  the  sodium 
sulphate  solution,  using  for  this  purpose  a  pipette  with  a 
long  tapering  point.  The  rubber  tube,  which  is  connected  to 
the  side  tube  of  the  proximal  limb  of  the  manometer,  is  to 
be  completely  filled  with  the  sulphate  solution,  taking  great 
care  to  see  that  all  air  is  expelled  from  the  tube  and  manom- 
eter. Close  the  clamp  on  the  tubing  which  connects  the 
rubber  tube  and  manometer.  With  a  short  piece  of  rubber 
tubing  connect  a  large  pipette  (25  cc. )  filled  with  the  sul- 
phate solution  to  the  upper  opening  of  the  proximal  limb  of 
the  manometer  and  blow  in  the  pipette  so  as  to  raise  the 


-  EXPERIMENTAL  PHARMACOLOGY.  201 


202  LABORATORY  GUIDE  IN 


BXPBRIMBNTAL  PHARMACOLOGY  203 

pressure  until  there  is  a  difference  of  10  or  12  cm.  in  the 
height  of  the  mercury  in  the  two  hmbs.^  Retain  this  pres- 
sure by  closing  the  piece  of  rubber  tubing  with  a  spring  or 
screw  clamp  and  disconnect  the  pipette. 

Connect  the  distal  end  of  the  rubber  tube  which  is  at- 
tached to  the  side  tube  of  the  manometer  to  the  carotid  can- 
nula and  open  the  clamps  so  as  to  allow  the  carotid  pulsa- 
tions to  be  transmitted  to  the  mercury.  Arrange  the  writing 
lever  so  that  it  will  record  the  pulsations  on  a  blackened 
drum  with  a  time  marker  writing  below  the  tracing.  Keep 
the  writing  lever  in  light  contact  with  the  drum  by  means 
of  a  small  weight  suspended  by  a  thread  from  a  support 
above  the  drum,  taking  care  that  the  thread  does  not  press 
too  hard  against  the  lever. 

Administer  the  following, drugs  in  the  manner  described, 
taking  a  short  piece  of  normal  tracing  before  each  is  given, 
and  waiting  until  the  effects  of  one  drug  have  passed  oft' 
Ijefore  applying  the  next.  Observe  the  precautions  as  tc 
the  injection  of  any  air. 

1.  Amyl  nitrite:  3  drops  on  absorbent  cotton  applied  to 
the  animal's  nose  or  to  the  tracheal  tube  if  that  has  been  in- 
serted. 

2.  Chloroform:  given  on  absorbent  cotton  as  above. 

3.  Ether:  given  on  absorbent  cotton. 

4.  Suprarenal  extract.  Adrenalin  chloride  (i-iooo)  ;  T 
drop  in  salt  solution  injected  into  the  vein.  Wash  the  can- 
nula and  syringe  thoroughly  before  injecting  the  next  drug. 

5.  Nicotine  chloride  i  mg.  diluted  with  salt  solution. 


^  The  pressure  may  also  be  raised  by  means  of  a  pressure  bottle  partially 
filled  with  sulphate  solution  and  suspended  from  the  ceiling.  A  tube  from 
this  bottle  may  be  connected  with  the  manometer  in  the  manner  described 
above. 


204  LABORATORY  GUIDB  IN 

6.  Pituitary  gland  extract  (Pituitrin  0.5  cc.)  in  salt  so- 
lution. 

7.  Digitalis,  i  cc.  of  the  tincture  from  which  the  alcohol 
has  been  evaporated  (given  intravenously). 

8.  Barium  chloride  20  mg.  in  salt  solution. 

Allow  the  drum  to  continue  rotating  a  short  distance  after 
the  heart  has  stopped,  so  that  the  writing  lever  traces  a 
straight  line  from  which  measurements  may  be  made  to 
ascertain  the  absolute  blood  pressure.  Remove  the  tracings 
from  the  drums  and  fix  them  by  passing  them  through  a 
solution  of  shellac  in  alcohol. 

The  blood  pressure  tracings  are  to  be  analyzed  according 
to  the  directions  given  on  page  215. 


EXPERIMENTAL  PHARMACOLOGY.  205 


2o6  LABORATORY  GUIDE  IN 


.EXPURIMUNTAL  PHARMACOLOGY 


207 


Effect  of  Drugs  on  the  Mammalian  Heart  and  Blood 
Pressure. 

Anaesthetize  a  dog  (page  12)  and  when  anaesthesia  is 
complete,  tie  the  animal  on  its  back  on  the  operating  table. 
Make  an  incision  through  the  skin  in  the  median  line  of  the 
i.ieck,  and  after  exposing  the  trachea  insert  a  cannula  in  the 
usual  way  (page  18).  Expose  a  vein,  either  one  of  the  jug- 
ulars or  a  saphenous  in  the  leg,  and  insert  a  venous  cannula. 

Also  dissect  out  an  artery,  either  one  of  the  carotids  or 
a  femoral  and  insert  a  cannula  into  it  and  arrange  it  for 
blood  pressure  tracing  according  to  the  method  described 
in  the  previous  experiment. 

Dissect  out  the  vagus  nerve  on  one  side,  tie  a  ligature 
around  it  and  cut  the  nerve  between  the  ligature  and  the 
head. 

Continue  the  skin  incision  in  the  median  line  from  the 
neck  down  the  thorax  to  an  inch  or  two  below  the  end  of  the 
sternum,  and  deepen  the  cut  to  the  bone.  Stop  all  bleeding 
points  with  artery  forceps. 

Connect  the  tubing  of  the  bellows  with  the  tracheal  tube 
and  start  artificial  respiration. 

Saw  through  the  entire  length  of  the  sternum,  keeping  ex- 
actly in  the  median  line  and  complete  the  division  with  bone 
forceps;  draw  the  two  sides  of  the  thorax  well  apart  and 
secure  them  to  the  sides  of  the  table  with  sharp  hooks. 

Regulate  the  amount  of  air  entering  the  lungs  by  means 
of  a  screw  clamp  placed  on  a  piece  of  rubber  tubing  on  the 
arm  of  the  tracheal  cannula. 

Note. — During  the  process  of  sawing  through  the  sternum  and  opening  the 
thorax  the  animal  must  be  very  deeply  under  the  anaesthetic,  otherwise  the 
heart  may  go  into  delirium. 


2o8 


LABORATORY  GUIDE  IN 


Cut  both  phrenic  nerves  as  they  pass  down  to  the  di- 
aphragm lying  in  plain  sight  on  either  side  of  the  pericardial 
sac. 

Open  the  pericardium  along  its  entire  length  with  a  pair 
of  scissors  and  fasten  it  to  the  cut  edges  of  the  sternum  by 
means  of  four  or  five  stitches. 


Fig.  No.   8.     IVlyocardiograph  for  dog's  heart. 

Take  a  stitch  in  either  side  of  the  right  ventricle  with 
small  curved  needles  and  thread,  and  tie  the  thread  into  the 
heart  in  the  same  way  as  you  did  with  the  turtle.  Similarly 
take  stitches  in  the  right  auricle.  If  bleeding  should  occur 
from  the  needle  passing  completely  through  the  wall  of 
either  chamber  take  up  the  bleeding  point  with  a  pair  of  dis- 
secting forceps  and  tie  a  ligature  around  it. 

Attach  the  two  sets  of  levers  of  the  dog's  myocardiograph^ 

*  The  dog  myocardiograph  (Fig.  8)  is  constructed  on  exactly  the  same  prin- 
ciples as  is  the  myocardiograph  for  the  turtle's  heart  (Fig.  7),  excepting 
that,  as  is  shown  in  the  figure,  it  is  double,  so  that  it  will  record  the  move- 
ments of  both  auricle  and  ventricle.  The  apparatus  has  other  modifications  as 
is  seen  in  the  plate,  but  the  principle  is  not  essentially  altered.  A  descrip- 
tion of  that  part  of  the  apparatus  which  is  connected  to  the  heart  is  found  in 
the  Jour,   of  Physiol.,   1897,   XXI,  P-   213. 


■  nXPURIMBNTAL  PHARMACOLOGY  209 


2IO 


LABORATORY  GUIDB  IN 


BXPERIMBNTAL  PHARMACOLOGY 


211 


tightly  (Fig.  8)  to  the  heart  by  means  of  the  threads  and 
arrange  the  writing  levers  to  record  the  cardiac  movements 
on  a  blackened  drum.  Arrange  a  time  marker  to  write  just 
below  the  cardiac  levers,  and  the  blood  pressure  writing  point 
at  a  convenient  location  on  the  drum  and  nearly  in  a  line 
with  the  levers. 


Fig.  No.  9.     Anaesthetic  inhaler  for  dogs.     By  rotating  handle   of  lever  either 
air  or  anaesthetic  may  be  forced  into  the  animal's  lungs. 

Start  the  kymograph  and  take  a  normal  tracing,  after 
which  take  tracings  to  show  the  effects  of  the  following: 

1.  Vagus  stimulation,  weak  and  strong  currents. 

2.  Chloroform  (given  with  special  inhaler.  Fig.  9)  ; 
continue  the  tracing  so  as  to  show  the  recovery. 

3.  Ether  (given  with  special  inhaler,  Fig.  9)  ;  continue 
the  tracing  so  as  to  show  the  recovery. 

4.  Suprarenal  extract.  (Adrenalin  chloride,  i-iooo), 
I  drop  added  to  4  cc.  salt  solution  and  injected  into  the  vein 
with  a  syringe. 

5.  Alcohol,  10  cc.  of  50%  given  intravenously. 

6.  Pituitary  gland  extract  (Pituitrin)  0.5  cc.  in  salt 
solution. 


212  LABORATORY  GUIDE  IN 

7.  Amyl  Nitrite.  3-5  minims  dropped  into  the  side  of 
the  tracheal  cannula. 

8.  Nicotine  chloride.    2  mg.  in  salt  solution. 

9.  Digitalis.  2  cc.  of  the  tincture  which  has  been  evap- 
orated to  one-half  volume  and  made  up  to  original  bulk 
with  salt  solution. 

10.  Digitalis.  Toxic  doses.  Give  3  cc.  doses  repeatedly 
until  the  animal  dies  allowing  about  4-minute  intervals  be- 
tween injections. 

Mark  the  tracings  carefully  so  that  they  can  all  be  identi- 
fied and  fix  them  in  shellac  as  before.  Analyze  them  ac- 
cording to  directions  given  on  page  219. 

Cut  off  the  head  of  the  animal  and  as  soon  as  convenient 
dissect  out  the  submaxillary  ducts  according  to  the  directions 
given  on  page  255.  This  dissection  will  allow  more  speedy 
operating  on  the  living  animal  in  the  experiment  on  the  sali- 
vary secretion. 


.nXPERIMENTAL  PHARMACOLOGY  213 


214  LABORATORY  GUIDE  IN 


.  nXPBRIMUNTAL  PHARMACOLOGY  215 

Analysis  of  Tracings. 

Graphic  records  have  been  obtained  in  three  experiments 
(turtle's  heart,  dog's  heart  and  blood  pressure)  and  it  is  of 
first  importance  that  these  records  should  be  carefully 
analyzed  in  order  to  properly  study  the  changes  induced  by 
the  various  drugs.  A  few  hints  are  given  below  to  call  at- 
tention to  some  of  the  more  important  points  to  be  looked 
for,  and  some  suggestions  are  made  as  to  the  best  way  of 
studying  the  tracings. 

Tracings   Obtained   from   the    Turtle's  Heart. 

It  is  rarely  necessary  in  these  records  to  take  accurate 
measurements,  as  the  changes  induced  by  the  drugs  are  gen- 
erally quite  marked. 

Describe  the  effect  of  stimulation  of  the  vagus,  noting 
whether  the  heart  stops  in  systole  or  diastole.  Likewise, 
describe  the  condition  of  the  heart  under  pilocarpine  (before 
and  after  atropine  administration)  and  also  the  effect  of  the 
atropine. 

Under  chloroform  note  especially  the  strength  of  the  heart 
as  indicated  by  the  extent  of  contraction  and  of  dilatation, 
which  is  measured  by  the  distance  of  the  tracing  from  the 
base  line. 

Summarize  the  actions  of  the  different  drugs. 

Tracings  from  the  Blood  Pressure  Experiment. 

Study  these  first  with  regard  to  changes  in  pressure.  Take 
careful  measurements  of  the  distance  of  the  tracing  from 
the  base  line  at  various  points  along  the  course  of  the  curve ; 


2i6  LABORATORY  GUIDE  IN 

viz.,  before  the  drug  is  injected  to  get  the  normal;  after  the 
action  of  the  drug  becomes  apparent ;  and  later  at  short  in- 
tervals of  time  which  will  depend  upon  the  drug  used. 
Where  the  curve  is  fairly  uniform  readings  every  thirty  sec- 
onds are  frequent  enough. 

Measuring  in  this  way  from  the  base  line  will  only  give 
relative  changes  in  pressure,  which  is  all  that  is  necessary  in 
these  experiments.  To  get  the  absolute  pressure,  the  zero 
line  which  was  marked  on  the  tracing  would  have  to  be  used, 
measuring  its  distance  above  or  below  the  base  line  and  add- 
ing or  subtracting  this  distance  from  the  figures  obtained 
above.  This  result  must  then  be  doubled  as  there  are  two 
arms  of  mercury  in  the  manometer. 

The  changes  in  the  rate  of  the  heart  are  studied  in  the 
following  manner:  Draw  two  lines  parallel  to  each  other 
and  perpendicular  to  the  base  line  so  that  they  shall  intersect 
the  tracing.  The  distance  of  the  lines  from  each  other  is 
determined  by  the  rate  of  the  drum  as  indicated  by  the  time 
marker;  they  are  usually  drawn  to  include  between  them  a 
space  equal  to  five  or  ten  seconds.  Count  the  heart  beats  in 
the  tracing  included  between  the  two  lines  and  estimate  the 
rate  of  the  heart  per  minute.  These  estimations  are  made 
along  the  curve  at  the  same  points  as  the  pressure  changes 
are  measured. 

Also  describe  any  other  changes  in  the  curves,  such  as  ir- 
regularities of  the  heart  or  of  the  respiration,  etc. 

Summarize  the  effects  of  the  different  drugs  upon  the 
pressure  of  the  blood  and  upon  the  rate  of  the  heart. 


EXPBRIMBNTAL  PHARMACOLOGY  217 


2i8  LABORATORY  GUIDE  IN 


.EXPERIMENTAL  PHARMACOLOGY  219 

Tracing  from  the  Dog's  Heart. 

These  are  to  be  studied  and  analyzed  by  a  combination  of 
the  methods  outlined  above.  The  rate  of  the  heart  is  ascer- 
tained as  described  under  the  directions  for  studying  the 
blood  pressure  tracings,  while  the  strength  of  the  organ  as 
indicated  by  the  extent  of  systole  and  diastole  is  measured 
from  the  base  line.  Describe  all  other  changes  in  the  trac- 
ings, and  in  your  notes  make  a  full  summary  of  the  actions 
of  the  different  drugs. 


220  LABORATORY  GUIDU  IN 

Digitalis  in  Auricular  Fibrillation. 

One  of  the  most  important  disturbances  of  the  heart  in 
which  digitaHs  is  employed  and  in  which  it  probably  exerts 
its  most  favorable  action  is  in  Auricular  Fibrillation.  In  this 
condition  there  is  no  orderly  contraction  in  the  auricle,  but 
due  perhaps  to  excessive  irritability  of  the  muscle  there  is 
a  constant  fibrillary  twitching  in  all  parts  of  the  auricular 
wall.  These  fibrillary  contractions  not  only  prevent  the 
auricle  from  emptying  itself  properly,  but  also  incessantly 
bombard  the  auricular-ventricular  bundle.  These  stimuli 
coming  to  the  bundle  so  rapidly,  only  a  certain  number  of 
them  can  get  through  and  some  of  these,  coming  at  irregular 
intervals,  will  strike  the  ventricle  when  it  is  in  its  refractory 
stage.  The  net  result  of  these  irregular  stimuli  upon  the 
ventricle  is  to  make  it  very  irregular  with  consequent  marked 
arhythmia  of  the  pulse.  At  first  these  attacks  of  auricular 
fibrillation  may  be  quite  transient  and  occur  at  long  in- 
tervals, but  later  on  they  may  be  more  frequent  and  grad- 
ually the  condition  becomes  permanent. 

In  this  form  of  cardiac  disturbance,  as  stated,  digitalis  is 
of  great  value  and  it  will  frequently  restore  the  ventricular 
rhythm  to  a  state  of  almost  complete  regularity.  This  may 
be  accomplished  either  by  an  effect  upon  the  ventricle  itself, 
altering  it  in  such  a  way  that  it  does  not  respond  so  readily 
to  the  rapid  and  irregular  stimuli  coming  down  from  the 
auricle  or  it  may  interfere  with  the  passage  of  the  stimuli 
through  the  Bundle  of  His  so  that  fewer  reach  the  ventricle. 
In  any  case  the  net  result  of  the  administration  of  digitalis 
is  a  disappearance  of  the  cardiac  arhythmia  and  an  im- 
provement in  the  circulation.     There  is  no  change  in  the 


■  BXPBRIMBNTAL  PHARMACOLOGY  221 


222  LABORATORY  GUIDE  IN 


MXPBRIMBNTAL  PHARMACOLOGY  223 

fibrillary  contractions  of  the  auricle  only  their  influence  on 
the  ventricle  is  removed. 

This  condition  of  Auricular  Fibrillation  can  be  produced 
experimentally  and  the  action  of  digitalis  upon  it  demon- 
strated. 

The  early  part  of  the  experiment  is  carried  out  and  the 
dog  prepared  in  the  same  manner  as  was  outlined  in  the 
work  upon  the  dog's  heart  (page  207).  After  the  carotid 
has  been  prepared  for  a  pulse  tracing  or  blood  pressure  and 
the  myocardiograph  has  been  attached  to  the  heart,  the  two 
wires  from  a  secondary  coil  are  attached  to  the  two  poles 
of  that  portion  of  the  myocardiograph  which  is  attached  to 
the  auricle,  and  a  weak  tetanizing  current  is  passed  through 
the  auricle.  The  chamber  responds  immediately  by  passing 
into  a  condition  of  fibrillation.  The  electric  current  must 
not  be  too  strong  or  it  may  spread  to  the  ventricle  and  set 
up  delirium  in  that  chamber  also.  When  a  satisfactory 
strength  of  current  is  found,  the  auricular  recording  lever 
should  show  merely  an  irregular  twitching  while  that  from 
the  ventricle  should  record  a  strong  but  very  irregular  con- 
traction. The  pulse  tracing  would  also  naturally  be  ir- 
regular. 

The  tincture  of  digitalis  (from  which  the  alcohol  has 
been  evaporated  and  its  original  volume  made  up  by  salt 
.solution)  may  now  be  injected  intravenously  in  3  cc.  doses, 
the  injections  not  being  repeated  at  shorter  intervals  than 
about  four  minutes.  After  a  few  of  these  injections  have 
been  given  in  the  manner  described,  the  heart  will  show  the 
effect  of  the  digitalis  in  a  strong  regular  beat  of  the  ventricle, 
regular  pulse,  and  probable  increase  in  blood  pressure.  The. 
auricle  will  still  show  its  fibrillary  contractions. 


224  LABORATORY  GUIDB  IN 

The  importance  of  the  action  of  digitalis  in  this  condition 
is  shown  by  the  fact  that  it  is  said  that  of  all  forms  of 
cardiac  arhythmias  auricular  fibrillation  constitutes  about 
40%. 


-EXPERIMENTAL  PHARMACOLOGY  225 


226  LABORATORY  GUIDE  IN 


BXPBRIMENTAL  PHARMACOLOGY  227 

Perfusion  of  Blood  Vessels. 

.The  experiment  can  be  carried  out  either  upon  the  kid- 
ney or  other  organ  such  as  a  spleen  removed  from  a  dog, 
cat  or  rabbit  or  upon  the  vessels  of  a  frog.  The  latter 
animal  proves  very  successful  and  that  method  will  there- 
fore be  described  first.  Select  a  fairly  large  frog  (30  G.- 
40  G.),  pith  it  and  expose  the  heart  and  insert  a  cannula  in 
one  aorta,  pointing  it  away  from  the  heart  including  the 
second  aorta  in  the  ligature  with  which  the  cannula  is  tied. 
Make  a  small  cut  in  the  sinus  venosum  so  that  the  fluid 
which  is  perfused  through  the  vessels  can  escape.  Suspend 
the  frog  by  its  jaw  and  place  under  it  a  receptacle  in  which 
the  outflow  can  be  caught.  About  a  foot  above  the  frog 
place  a  small  pressure  bottle  (500  cc),  the  lower  opening 
of  which  is  fitted  with  a  piece  of  rubber  tubing  upon  which 
is  a  snap  clamp.  Fill  the  aortic  cannula  with  Locke's  solu- 
tion or  physiological  salt  and  fill  the  pressure  bottle  about 
half  full  of  the  same  solution.  When  all  the  air  is  excluded 
from  the  rubber  tubing  connect  it  with  the  aortic  cannula. 
Open  the  snap  clamp  which  has  been  placed  on  the  rubber 
tube  and  allow  the  perfusion  to  proceed,  measuring  the  out- 
flow every  five  minutes.  After  about  three  readings  there 
should  be  a  fairly  uniform  outflow  and  200  mg.  of  sodium 
nitrite  are  added  to  the  fluid  in  the  bottle  and  the  solution 
mixed  thoroughly.  The  readings  of  the  outflow  are  con- 
tinued and  after  one  or  two  the  amount  obtained  should 
be  very  considerably  increased.  If  it  is  not  increased  within 
a  reasonable  time  further  nitrite  may  be  added.  Now  re- 
place the  nitrite  solution  in  the  flask  with  fresh  salt  solu- 
tion and  wash  out  the  frog's  vessels,  seeing  whether  the 


228  LABORATORY  GUIDE  IN 

outflow  will  return  to  normal.  After  two  or  three  readings 
add  to  the  solution  in  the  flask  i  cc.  of  a  i-iooo  solution  of 
adrenalin.  Continue  the  readings  until  a  marked  decrease 
in  outflow  is  obtained.  In  place  of  the  adrenalin  tincture 
of  digitalis  in  the  strength  of  3  cc.  of  tincture  to  100  cc.  of 
salt  solution  may  be  used. 

If  a  kidney  or  other  organ  is  to  be  used  in  place  of  the 
frog's  vessels  the  experiment  is  carried  out  in  essentially  the 
same  manner. 

The  kidneys  which  have  been  removed  from  a  dog,  cat 
or  rabbit  should  not  be  kept  any  longer  than  is  absolutely 
necessary  after  being  removed  from  the  animal,  certainly 
not  more  than  twenty-four  hours,  as  after  that  time  post- 
mortem changes  have  advanced  so  far  as  to  interfere  with 
the  action  of  drugs  on  the  vessel  walls. 

In  preparing  the  kidneys  for  the  experiment  it  is  usually 
best  not  to  remove  the  capsule,  as  in  so  doing  the  paren- 
chyma is  frequently  torn.  Remove  the  fat  and  connective 
tissue  from  around  the  vessels  and  insert  a  glass  cannula  in 
the  artery.  Fill  the  artery  and  cannula  with  salt  solution. 
Fasten  a  small  glass  reservoir  (say  250  cc.)  about  1.5  meters 
above  the  table  and  connect  a  long  piece  of  rubber  tubing 
with  its  lower  orifice,  and  fill  reservoir  and  tube  with  Locke's 
solution  and  clamp  the  tube. 

Connect  the  lower  end  of  the  rubber  tube  with  the  arterial 
cannula  taking  care  that  all  air  is  expelled.  Remove  the 
clamp  from  the  tubing  and  place  a  receptacle  under  the 
kidney  to  catch  the  fluid  after  it  has  passed  through  the 
vessels  and  continue  the  experiment  as  described  above  for 
occasions  where  a  frog  is  used. 


nXPBRIMBNTAL  PHARMACOLOGY  229 


230  LABORATORY  GUIDE  IN 


.EXPERIMENTAL  PHARMACOLOGY  231 

What  do  the  changes  in  the  rate  of  outflow  under  the 
two  drugs  indicate  in  regard  to  their  action  on  the  blood 
vessels?  How  do  the  results  help  to  explain  the  observa- 
tions made  in  the  experiment  on  blood  pressure  (page  200). 


232  LABORATORY  GUIDE  IN 

Action  of  Drugs  on  Isolated  Tissues. 

The  action  of  many  drugs  in  the  body  may  be  studied  to 
advantage  by  observing  the  effects  of  these  drugs  upon  iso- 
lated tissues  or  organs,  which  during  the  time  that  they  are 
in  use  are  under  as  nearly  normal  conditions  as  possible  as 
far  as  temperature  and  oxygen  supply  are  concerned.  Those 
structures  which  are  most  commonly  employed  in  this  work 
are  the  uterus,  or  strips  of  intestine  or  blood  vessels. 

While  more  or  less  complicated  apparatus  is  needed  if 
quantitative  results  are  desired,  yet  if  the  requirements  are 
only  qualitative  quite  a  simple  arrangement  is  all  tha:t  is 
demanded.  The  Harvard  muscle  warmer  and  heart  lever, 
kymograph,  oxygen  or  air  supply,  and  warm  Locke's  solu- 
tion will  answer  all  purposes. 

In  case  the  uterus  is  to  be  used,  a  guinea  pig  (or  rabbit 
or  cat)  weighing  200  G.  or  300  G.  is  killed  by  a  blow  on 
the  head  and  the  abdomen  opened  and  uterus  exposed  and 
a  fine  thread  tied  around  the  ovarian  end  of  one  horn.  After 
the  organ  is  freed  from  the  posterior  wall,  another  thread 
is  tied  around  it  near  its  lower  end,  and  it  is  then  removed 
from  the  body.  Using  the  thread  on  the  vaginal  end  of  the 
organ,  the  uterus  is  now  tied  to  the  end  of  the  bent  metal 
rod  of  the  muscle  warmer.  The  thread  on  the  ovarian 
end  is  passed  through  the  round  opening  in  the  plate  disc 
and  fastened  to  a  muscle  lever  so  that  the  lever  will  record 
the  contractions  of  the  uterus  upon  a  lightly  smoked  drum. 
Uteri  differ  considerably  in  activity  as  some  are  so  active 
that  it  is  necessary  to  weight  the  lever  in  order  to  diminish 
the  extent  of  the  contractions  while  other  organs  are  quies- 


nXPHRIMBNTAL  PHARMACOLOGY  233 


234  LABORATORY  GUIDE  IN 


BXPBRIMENTAL  PHARMACOLOGY  '235 

cent  unless  aroused  to  action  by  some  drug.  The  lower 
end  of  the  glass  tube  of  the  muscle  lever  is  fitted  with  a 
piece  of  rubber  tubing  which  can  be  closed  by  a  snap  clamp, 
and  the  tube  is  then  adjusted  in  place  around  the  piece  of 
uterus,  and  filled  with  Locke's  solution  at  a  temperature  of 
38°  C.  Air  or  oxygen  is  led  to  the  bottom  of  this  tube  by 
means  of  a  capillary  glass  tube  or  a  fine  rubber  catheter. 
For  long  experiments  or  for  any  exact  work  the  muscle 
warmer  should  be  enclosed  in  an  outer  bath  of  water  main- 
tained at  body  temperature,  but  for  short  experiments  this 
is  hardly  necessary  as  the  Locke's  solution  is  replaced  at 
short  intervals,  after  the  use  of  each  drug,  by  fresh  solu- 
tion of  the  proper  temperature.  When  the  lever  is  record- 
ing properly  and  the  drum  is  moving  at  a  slow  rate,  the 
actions  of  the  following  drugs  may  be  studied  by  adding 
them  in  turn  to  the  solution  bathing  the  uterus.  When  the 
climax  of  the  action  is  reached,  the  used  solution  should 
be  drawn  off  and  replaced  by  fresh  and  the  uterus  allowed 
at  least  five  minutes  to  recover  before  another  drug  is  added. 

Add  to  the  solution  bathing  the  uterus  i  drop  of  adrenalin 
(i-iooo).  When  the  maximum  effect  is  obtained,  draw  off 
the  solution  and  replace  it  with  fresh  warm  solution  as 
directed  above.  After  giving  the  organ  a  rest,  add  i  or  2 
mg.  of  pilocarpine  and  then  immediately  after  putting  fresh 
Locke's  solution  on  the  uterus  give  i  to  2  mg.  Atropine 
sulphate.  This  may  be  followed  in  turn  by  pilocarpine 
again  ;  Fl.  Ext.  Ergot  0.3  cc. ;  Pituitary  Extract  (Pituitrin 
2  drops)  ;  each  in  a  fresh  solution. 

The  doses  given  above  may  have  to  be  modified  to  suit 
the  requirements  of  the  individual  organ.     When  the  ex- 


236  LABORATORY  GUIDE  IN 

periment  is  completed,  fix  the  tracings  with  shellac  and  later 
study  them  carefully,  taking  up  the  action  of  the  individual 
drugs,  comparing  the  results  with  those  obtained  with  these 
drugs  m  other  experiments. 


BXPERIMBNTAL  PHARMACOLOGY  237 


238  LABORATORY  GUIDE  IN 


EXPERIMENTAL  PHARMACOLOGY  239 

Diuresis. 

Anaesthetize  a  rabbit  with  paraldehyde  (page  11)  and 
when  anaesthesia  is  complete  tie  the  animal  on  the  operating 
board.  Cut  the  hair  from  the  neck  and  also  from  the  lower 
part  of  the  abdomen.  Expose  a  jugular  vein  and  insert  a 
cannula  as  usual. 

To  insert  a  cannula  in  the  bladder  to  collect  the  urine, 
make  an  incision  about  an  inch  and  a  half  long  in  the  median 
line  of  the  abdomen,  beginning  the  incision  at  the  symphysis 
and  extending  it  upward  the  distance  named.  Deepen  the 
cut  until  the  peritoneal  cavity  is  opened.  The  bladder  is 
usually  found  partially  distended  and  lying  amid  folds  of 
fat.  DraAV  it  out  through  the  opening  made  and  empty  it  by 
gentle  pressure,  catching  the  urine  in  a  dish  as  it  flows  from 
the  urethra.  Save  this  urine  to  be  tested  for  the  presence  of 
sugar. 

Tie  a  knot  loosely  in  a  piece  of  thread  and  lay  it  on  the 
abdomen  so  that  the  knotted  thread  will  encircle  the  bladder 
and  be  ready  to  tie  the  cannula  in  place  when  the  latter  shall 
have  been  inserted. 

After  locating  the  position  of  the  ureters,  so  as  to  avoid 
including  them  in  the  ligature,  take  hold  of  the  upper  sur- 
face of  the  bladder  on  the  two  sides  with  two  pairs  of  for- 
ceps and  draw  them  gently  up  and  outwards  so  as  to  exert  a 
slight  tension  on  the  organ.  While  one  operator  holds  the 
bladder  in  this  way  the  other  operator  makes  a  cut  with  a 
sharp  pair  of  scissors  midway  between  the  forceps  in  the 
fundus  of  the  bladder.  The  cut  will  vary  with  the  size  of 
the  cannula  flange  (a,  Fig.  10)  and  will  probably  average 
about  two   centimeters   in   length.     Into  this   incision  now 


240 


LABORATORY  GUIDE  IN 


place  the  cannula  so  that  its  flange  shall  be  entirely  sur- 
rounded by  bladder  tissue  and  tie  it  in  place  with  the  loose 
ligature  already  mentioned.  Examine  it  after  it  is  tied  to 
see  that  the  ureters  are  not  included. 

Fill  the  bladder  and  cannula  with  warm  physiological  salt 
solution  and  connect  the  end  of  the  cannula  with  the  piece 
of  bent  tubing  (b,  Fig.  10)  which  can  now  rest  on  a  support 
so  as  to  extend  over  the  end  of  the  op- 
erating board  where  small  weighed' 
dishes  are  to  be  placed  to  catch  the 
urine. 

As  soon  as  the  urine  begins  to  drop 
from  the  tube  a  record  of  the  time  is  to 
be  made,  and  thereafter  to  the  end  of 
the  experiment,  every  five  minutes,  the 
dish  with  the  urine  is  to  be  replaced 
by  a  clean  receptacle  and  the  first  one 
weighed,  and  the  weight  of  urine  re- 
corded. 

During  the  course  of  the  experiment 
the  animal  should  be  kept  covered  with 
a  towel  or  other  cloth  in  order  to  re- 
tain as  much  of  its  body  heat  as  possi- 
ble. 

Take  two  or  three  readings  until  a 
fair  regularity  is  obtained,  and  after 
this  normal  is  ascertained  inject  into  the 

Fig.    No.    io.      Bladder 
Cannula.      A.    Flange    to    Vein  40  Ulg.  of  thcocill^  dlsSolvcd  in  hot 
be    tied    in    bladder.      B. 
Bent    delivery    tube.  watCr. 


" 


^Theocin  is  artificial  theophyllin.  A  like  quantity  of  caffeine  might  be  given 
in  place  of  the  theocin  except  that  the  action  of  the  caffeine  lasts  much  longer 
and  may  prolong  the  experiment  unduly. 


.  BXPBRIMBNTAL  PHARMACOLOGY  241 


2^2  lABORA'tORY  GUIDH  IN 


nXPBRIMBNTAL  PHARMACOLOGY  243 

After  this  injection  the  urine  will  probably  be  increased 
and  the  next  drug  must  not  be  injected  until  the  flow  has 
returned  to  about  the  normal. 

Test  some  of  the  urine  which  was  in  the  bladder  at  the  be- 
ginning of  the  experiment  for  sugar  by  means  of  the  Fehling 
test.  If  none  is  found  make  one  or  tw^o  tests  later  in  the 
course  of  the  experiment  with  urine  which  is  excreted  under 
the  action  of  the  theocin  or  sodium  nitrate  until  sugar  shall 
be  found. 

The  excretion  of  iodide  in  the  urine  may  also  be  studied 
by  the  careful  injection  intravenously  of  30  mg.  sodium 
iodide.  The  urine  passed  later  is  tested  for  iodide  as  fol- 
lows :  add  a  few  drops  of  concentrated  sulphuric  acid  to 
the  urine  to  be  tested,  and  to  this  some  10%  sodium  nitrite 
solution  and  some  starch  paste  which  if  iodide  is  present 
sliould  be  colored  blue. 

When  the  amount  of  urine  has  fallen,  inject  0.250  G. 
(5  cc.  of  a  5%  solution)  of  sodium  nitrate  and  collect  the 
urine  as  before. 

Note. — If  time  allows,  the  diuretic  effect  of  digitalis  may  be  studied  in  the 
same  way. 

Caffeine,  sodium  nitrate  and  digitalis  are  taken  as  repre- 
sentatives of  the  large  class  of  drugs  known  as  Diuretics 
from  their  action  in  increasing  the  amount  of  urine. 

They  may  act  either  on  the  heart  or  on  the  kidney,  and 
they  are  known  as  cardiac  or  renal  diuretics  according  to 
the  point  of  action.  Digitalis  is  an  example  of  the  former 
class,  as  it  causes  diuresis  indirectly  through  its  effects  on 
the  circulation.  Caffeine  probably  acts  directly  on  the  renal 
epithelium,  while  various  salts,  of  which  sodium  nitrate  is 
an  example,  induce  diuresis  by  their  salt  action  (sahne 
diuretic)  in  addition  to  which  there  may  be  an  action  on  the 
kidney  epithelium. 


244  LABORATORY  GUIDE  IN 

Therapeutic  uses  of  the  Diuretics. 

See  also  under  Caffeine  and  Digitalis,  Therapeutic  uses, 
(pages  140  and  179). 

These  drugs  or  their  allies  are  chiefly  employed  in  diseased 
conditions  in  which  the  amount  of  urine  excreted  is  below 
the  normal,  a  condition  which  may  result  from  disease  of 
the  kidneys  or  of  the  heart.  In  conditions  of  oedema  or 
ascites,  there  is  an  abnormal  collection  of  fluid  in  the  tis- 
sues; its  removal  may  be  aided  by  the  use  of  the  diuretics. 
In  poisoning  with  an  irritant  drug  which  is  eliminated  by 
the  kidneys,  the  latter  may  be  protected  from  the  action  of 
the  toxic  substance  by  increasing  the  volume  of  urine 
through  the  administration  of  these  drugs. 


nXPBRIMBNTAL  PHARMACOLOGY  245 


246 


LABORATORY  GUIDE  IN 


-  BXPBRIMENTAL  PHARMACOLOGY  247 

Action  of  Drugs  on  the  Cervical  Sympathetic  Nerves  and 
on  Intestinal  Peristalsis.^ 

Ansesthetize  a  rabbit  (an  albino  if  possible)  with  paralde- 
hyde (page  11)  and  when  anaesthesia  is  complete  tie  the 
animal  on  the  operating  board  and  remove  the  hair  from 
the  neck  region.  Expose  the  jugular  vein  on  one  side  and 
also  the  trachea  and  insert  cannulas  in  each.  Expose  the 
carotid  sheath  on  the  other  side,  open  it,  laying  bare  the 
vessels  and  nerves.  The  largest  nerve  seen  is  the  vagus  and 
the  smallest  is  the  depressor  nerve,  while  the  third  is  the 
cervical  sympathetic  trunk,  which  is  to  be  separated  very 
carefully  from  all  connective  tissues  and  a  fine  thread  passed 
under  it.  Make  sure  you  have  the  correct  nerve  by  gently 
raising  it  and  stimulating  it  with  a  weak  tetanizing  current, 
observing  meantime  the  effect  on  the  pupil  on  the  corre- 
sponding side;  it  should  dilate  during  the  stimulation.  Tf 
the  correct  nerve  has  been  isolated  tie  the  ligature  around  it 
as  low  down  in  the  neck  as  convenient  and  cut  the  nerve 
trunk  below  the  ligature.  Hold  the  animal's  ears  up  gently 
against  the  light  so  that  the  vessels  may  be  clearly  seen,  and 
observe  the  difference  in  their  size  on  the  two  sides ;  note 
also  the  difference  in  the  temperature  of  the  two  ears.  The 
differences  in  the  vessels  in  the  two  ears  may  be  best  seen 
by  comparing  the  smaller  branches  rather  than  the  main 
arteries.  Compare  the  size  of  the  pupils  on  the  two  sides. 
How  would  you  explain  these  changes  ? 

Make  a  longitudional  incision  about  10  cm.  long  in  the 
middle  line  of  the  abdomen  extending  the  cut  completely 


1  The  expjeriment  on  intestinal  peristalsis  may  be  run  as  a  separate  experi- 
ment (see  page  292)  or  as  directed  here  in  conjunction  with  the  cervical 
=;-vmpathetic   experiment. 


248  LABORATORY  GUIDE  IN 

through  the  wall.  Pulling  the  two  sides  of  the  wall  apart 
insert  in  the  incision  a  glass  evaporating  dish  measuring 
about  10  cm.  in  diameter.  Place  the  dish  between  the  skin 
and  muscle  layers  so  that  it  will  be  held  in  place  by  the 
wall  and  thus  serve  as  a  window.  It  may  be  necessary  to 
pull  the  caecum  to  one  side  in  order  to  permit  of  a  free 
view  of  the  small  intestine.  Observe  closely  the  normal 
movements  of  the  intestine. 

Now  raise  the  trunk  of  the  cut  cervical  nerve  carefully  by 
the  ligature  and  stimulate  it  with  the  electric  current  as  be- 
fore. During  this  stimulation  look  especially  for  any 
changes  in  the  calibre  of  the  ear  vessels  on  that  side;  and 
for  any  changes  in  the  position  of  the  eyeball,  or  of  the  eye- 
lids, or  in  the  size  of  the  pupil.  After  the  changes  have  been 
seen  and  described,  inject  into  the  jugular  vein  3  mg.  of  nico- 
tine chloride.  Immediately  after  the  injection  notice  the  ef- 
fect of  the  drug  on  the  animal's  respiration  (make  sure  the 
animal  starts  breathing  again,  otherwise  start  artificial  res- 
piration), and  also  look  for  any  movement  in  the  anim^al's 
whiskers. 

Again  stimulate  the  cut  cervical  sympathetic  comparing 
'  the  results  with  those  obtained  at  the  first  stimulation. 
(Should  they  be  the  same  as  before,  it  will  be  necessary  to 
inject  more  nicotine.)  Now  using  very  careful  dissection 
trace  the  trunk  of  the  sympathetic  toward  the  head  until  the 
superior  cervical  ganglion  is  reached.  It  will  be  recognized 
as  a  small  whitish  enlargement  on  the  trunk  of  the  nerve. 
When  it  is  found,  stimulate  the  trunk  of  the  sympathetic  be- 
yond the  ganglion,  that  is,  between  the  ganglion  and  the 
head  of  the  animal.  Compare  the  results  of  stimulation  at 
this  point  with  those  obtained  below  the  ganglion. 


EXPERIMENTAL  PHARMACOLOGY  249 


250  LABORATORY  GUIDE  IN 


BXPBRIMENTAL  PHARMACOLOGY  251 

Note. — In  nddition  to  the  nerve  fibres  which  run  to  the  eye  by  way  of  the 
cervical  sympathetic  and  which  are  distributed  to  the  radial  mtiscle  fibres  of 
the  iris  there  are  nerves  which  control  the  circular  fibres.  These  come  from 
the  motor  oculi  to  the  ciliary  ganglia  and  run  from  that  point  to  the  iris. 
The  size  of  the  pupil  can  be  changed  by  drugs  acting  along  the  course  of 
these  fibres  also. 

Inject  into  the  jugular  vein  5  mg.  of  pilocarpine  (i  cc.  of 
0.5%  solution)  and  observe  any  change  in  the  pupil  and  also 
in  intestinal  peristalsis.  (Note  any  effect  on  the  heart  rate.) 
Now  inject  i  mg.  atropine  sulphate  (i  cc.  of  0.1%  solution). 
What  effect  has  it  upon  the  pupil,  peristalsis  and  heart  rate? 

Finally  inject  a  small  dose  of  suprarenal  extract 
(adrenalin  i-iooo  solution,  i  drop  in  salt  solution)  note 
changes  in  peristalsis,  calibre  of  vessels  and  heart  rate. 

What  effect  has  nicotine  upon  the  sympathetic  gangha? 

Cocaine  dilates  the  pupil  by  stimulating  the  terminations 
cf  the  nerves  from  the  superior  cervical  ganglia  which  end 
on  the  radial  fibres. 

Physostigmine  (Eserine)  acts  on  the  same  point  and  in 
the  same  manner  as  pilocarpine,  i.  e.,  on  the  nerve  termina- 
tions on  the  circular  muscular  fibres;  atropine  acts  at  the 
same  place,  but  instead  of  stimulating  the  nerve  endings,  its 
action  is  to  paralyze  them,  removing  the  pilocarpine  effects. 

Therapeutic  Uses. 

Physostigmine  (eserine)  and,  less  commonly,  pilocarpine 
are  used  in  ophthalmological  practice  to  contract  the  pupil  of 
the  eye  in  certain  diseased  conditions,  the  most  important  of 
which  is  glaucoma.  For  this  purpose  a  solution  of  ^  to  1% 
is  dropped  in  the  eye  as  often  as  necessary.  Physostigmine 
is  also  occasionally  used  to  stimulate  intestinal  peristalsis  in 
cases  of  atony  of  the  intestine.  For  this  purpose  it  is  given 
by  hypodermic  injection. 


252  LABORATORY  GUIDB  IN 

Atropine  is  used  to  dilate  the  pupil  to  permit  of  ophthal- 
moscopic examinations,  and  as  a  therapeutic  agent  in  many 
ocular  diseases.  It  is  also  used  in  cases  of  colic  or  griping 
to  overcome  the  abnormal  contractions  of  the  intestine. 
Homatropine,  an  artificial  alkaloid  resembling  atropine  in 
its  action  on  the  eye,  is  largely  used  as  a  substitute  for  the 
latter,  as  its  action  is  not  nearly  so  prolonged. 

Cocaine  is  also  largely  employed  to  dilate  the  pupil  to 
allow  of  examination  of  the  interior  of  the  eye. 

Mydriatics  are  drugs  which  dilate  the  pupil;  myotics  con- 
tract it. 


EXPERIMENTAL  PHARMACOLOGY  2 


JO 


254  LABORATORY  GUIDE  IN 


BXPBRIMBNTAL  PHARMACOLOGY  255 

Salivary  and  Pancreatic  Secretion.^ 

Ansesthetize  a  large  dog  with  morphine  and  chloretone 
(page  12)  and  if  necessary  supplement  them  by  the  use  of 
chloroform.  When  anaesthesia  is  complete,  expose  a  vein 
in  each  leg  and  insert  cannulas.  As  a  matter  of  precaution, 
the  trachea  may  also  be  exposed  and  a  cannula  tied  in  it  to 
permit  of  artificial  respiration  should  it  become  necessary. 

The  submaxillary  duct  and  chorda  tympani  nerve  are  to 
be  dissected  out  according  to  the  following  directions.  (This 
dissection  _  should  have  been  previously  carried  out  on  the 
head  of  a  dead  animal  (from  Exp.,  page  212)  so  as  to  allow 
greater  speed  in  operating  on  the  living  dog.) 

"Make  an  incision  3  or  4  inches  long  through  the  skin  and 
platysma  muscle  along  the  inner  border  of  the  lower  jaw, 
beginning  about  the  angle  of  the  mouth  and  continuing  back- 
ward towards  the  angle  of  the  jaw.  Ligate  doubly  and 
divide  any  vessels  that  come  in  your  way.  Divide  the  digas- 
tric muscle  about  its  anterior  third  and  clear  it  from  its  at- 
tachment. The  broad-,  thin  mylo-hyoid  will  now"  be  seen 
with  its  motor  nerve  lying  on  it.  Divide  the  muscle  across 
its  middle  and  dissect  it  up  carefully.  The  lingual  nerve  is 
seen  emerging  from  under  the  ramus  of  the  jaw,  running, 
transversely  towards  the  middle  line  and  then  passing  for- 
ward parallel  to  the  larger  hypoglossal  nerve.  In  its  trans- 
verse course  it  crosses  the  submaxillary  and  sublingual 
ducts.  The  chorda  tympani  leaves  the  lingual  nerve  and 
runs  backward  along  the  duct  towards  the  gland.  Put  a 
glass  cannula  into  the  duct  in  the  same  way  as  you  would 


^  The  salivary  and  pancreatic  secretion  experiments  may  be   run  separately   if 
preferred. 


256 


LABORATORY  GUIDE  IN 


put  a  cannula  in  a  vein.  Trace  back  the  lingual  and  put  a 
ligature  around  it  and  tie  it  as  far  back  as  possible,  and  then 
cut  it  centrally  so  as  to  permit  of  the  chorda  tympani  being 
put  on  electrodes."  (Stewart.)  Connect  the  cannula  with 
a  long,  horizontal  tube  on  which  a  scale  is  marked. 


Fio.  No.  II.     Dog's  head  dissectpd  to  show  Chorda  Tympani.     A.  Chorda  Tym- 
pani, B.  Wharton's  duct,  C.  Hypoglossal  nerve,  D.  Mylo-hyoid  muscle 
turned  back,  E.  Lingual  nerve,  F.  Digastric  muscle  cut,  G.  Ra- 
mus  of  jaw   cut   away   to   show   nerve   emerging   from 
behind  it. 


While  the  dissection  of  the  duct  is  being  carried  on,  a 
second  operator  is  to  dissect  out  the  cervical  sympathetic 
trunk^  on  the  same  side.  In  the  dog  the  cervical  sympathetic 
is  contained  in  the  carotid  sheath  and  closely  connected  with 
the  vagus,  from  which  it  may  be  separated  by  careful  dis- 


^  This  part  of  the  experiment  may  be  omitted  if  desired. 


EXPBRIMMNTAL  PHARMACOLOGY  257 


2^8  LABORATORY  GUIDB  IN 


EXPERIMENTAL  PHARMACOLOGY  259. 

section,  as  the  fibres  run  in  distinct  strands.  For  some  dis- 
tance below  the  superior  cervical  ganglion  the  two  nerve 
trunks  are  not  connected.  After  the  sympathetic  is  isolated 
tie  a  ligature  around  it  and  cut  the  nerve  below  it. 

If  the  experiment  has  been  properly  done,  both  nerves 
(viz.,  the  chorda  and  the  sympathetic)  can  be  stimulated  by 
placing  them  on  electrodes,  and  any  secretion  from  the  gland 
will  pass  into  the  horizontal  tube,  Avhere  it  can  be  measured 
on  the  scale. 

The  pancreatic  duct  is  exposed  in  the  following  manner. 
An  incision  is  made  through  the  abdominal  wall  about  6  or 
S  cm.  to  the  right  of  the  median  line  and  starting  from  the 
costal  border  and  extending  caudalwards  for  a  distance  of 
about  15  to  20  cm.  The  duodenum  is  located  and  pulled 
out  through  the  incision  and  the  pancreas  is  found  attached 
to  it  for  some  distance.  Starting  at  the  point  where  the 
tail  of  the  pancreas  leaves  the  intestine  and  following  up  to- 
ward the  pylorus  for  about  3  cm.  the  pancreas  is  separated 
from  its  anterior  attachment  to  the  intestine  by  blunt  dissec- 
tion. At  about  this  point  the  large  duct  of  the  pancreas  will 
be  found  entering  the  wall  of  the  intestme.  It  is  very  short. 
The  surrounding  tissue  is  separated  from  the  duct  and  a 
ligature  placed  around  it.  A  V-shaped  cut  is  made  in  it  and 
a  glass  cannula  inserted  and  fastened  by  means  of  the  liga- 
ture. A  small  calibre  tube  is  now  connected  to  the  cannula 
by  means  of  a  piece  of  rubber  tubing  and  the  pancreatic 
secretion  observed  either  by  means  of  a  scale  back  of  the 
tube  or  by  the  drops  falling  from  the  end.  The  intestinal 
loop  is  now  returned  to  the  abdomen  or  may  be  secured  out- 
side protected  by  warm  moist  towels  and  the  abdominal  in- 
cision closed  as  far  as  possible. 


26o  LABORATORY  GUIDE  IN 

1.  Stimulate  the  chorda  tympani  with  a  weak  induction 
current  and  watch  the  effect  on  the  secretion. 

2.  When  the  effect  of  chorda  stimulation  has  passed  off 
stimulate  the  sympathetic  trunk  (peripheral  or  head  end) 
and  observe  the  difference  in  the  quantity  and  quality  of  the 
saliva  when  compared  with  the  "chorda  saliva." 

3.  Inject  5  mg.  of  nicotine  chloride  and  observe  closely 
the  effect  upon  the  salivary  secretion.  Watch  the  effect 
upon  the  respiration  and  heart  rate.  (Be  sure  the  respira- 
tion continues  after  the  nicotine  has  been  injected.) 

4.  Stimulate  the  chorda  again  and  compare  the  results 
v/ith  those  obtained  in  i. 

5.  Stimulate  the  sympathetic  and  compare  with  2. 

6.  If  the  nerves  are  still  active,  repeat  the  injections  of 
nicotine,  giving  20  mg.  each  time  until  no  further  effect  is 


Preparation  of  secretin.  The  secretin  which  is  to  be  used  in  the  experi- 
nitni.  is  to  be  prepared  from  the  intestine  of  one  of  the  dogs  which  has  been 
used  earlier  in  the  course.  Its  mode  of  preparation  is  as  follows:  Immediately- 
after  the  animal  has  been  killed,  open  the  abdomen  and  remove  about  1.5 
meters  of  the  upper  part  of  the  intestine.  Cut  this  piece  open  longitudinally 
under  running  cold  water  and  after  it  is  cleansed,  spread  it  flat  on  a  piece 
of  glass  and  scrape  off  the  mucosa  by  means  of  a  second  piece  of  glass.  Put 
this  material  in  a  large  mortar  with  about  an  equal  volume  of  fine  clean 
sand  and  from  100  to  300  cc.  of  0.4%  HCl,  the  amount  depending  upon  the 
bulk  of  sand  and*  mucosa.  Grind  this  mass  now  thoroughly  for  ten  minutes 
sc  as  to  get  the  tissue  cells  well  broken  up.  Transfer  the  mixture  to  a  large 
evaporating  dish  and  boil  it  for  a  minute  or  two,  keeping  it  well  stirred.  Now 
add  a  sufficient  quantity  of  a  fairly  strong  solution  of  NaOH  so  as  to  give 
an  alkaline  reaction  and  follow  this  with  sufficient  25%  glacial  acetic  acid  to 
give  a  distinct  acid  reaction.  Strain  the  mixture  now  through  muslin,  pressing 
out  all  the  fluid  possible.  This  fluid,  which  should  be  rather  strongly  opalescent, 
is  now  partially  sterilized  by  placing  the  flask  containing  it  over  a  water  bath 
for  ten  minutes  and  then  plugging  it  with  absorbent  cotton.  Keep  it  in  a 
cool  place  until  it  is  needed.  It  may  be  injected  in  this  strength  or  still  bet- 
ter diluted  with  salt  solution.  The  normal  pouring  out  of  secretin  into  the 
blood  may  perhaps  be  best  simulated  by  diluting  the  stock  solution  of  secretin 
with  many  times  its  volume  of  physiological  salt  solution  and  then  placing  this 
solution  in  a  burette  and  connecting  it  with  a  femoral  vein,  allowing  it  to 
enter  the  blood  stream  slowly,  and  continuously.  Marked  effects  on  the  blood 
pressure  are  thus  avoided  and  a  constant  secretion  from  the  pancreas  obtained. 
This   latter   method   is   the   one   to   be   used   in   this   experiment. 


nXPERIMENTAL  PHARMACOLOGY  261 


262  LABORATORY  GUIDE  IN 


MXPBRIMBNTAL  PHARMACOLOGY  263 

obtained  on  nerve  stimulation.    How  do  the  effects  of  sub- 
sequent injections  compare  with  the  primary  injections? 

7.  Trace  the  trunk  of  the  sympathetic  to  the  superior 
cervical  ganglion  and  stimulate  the  nerve  beyond,  observing 
if  secretion  can  be  obtained. 

Inject  the  following  drugs  in  the  order  named,  observing 
their  effects  upon  both  the  salivary  and  pancreatic  secretion : 

8.  Pilocarpine,  5  mg. 

9.  Atropine,  i  mg. 

10.  Pilocarpine,  5  mg.,  compare  with  8. 

11.  Suprarenal   extract,  Adrenahn  chloride,   i-iooo  sol., 
5  drops  in  salt  solution. 

What  effect  has  nicotine  upon  the  sympathetic  ganglia? 
Compare  also  the  experiment  on  the  sympathetic  ganglia  in 
the  rabbit  (page  247). 

As  was  shown  in  this  experiment,  the  salivary  gland  is 
innervated  from  two  sources,  both  of  which  are  acted  on  by 
nicotine.  Pilocarpine  and  atropine  aft'ect  the  salivary  secre- 
tion by  acting  on  the  cranial  nerve  supply  of  the  gland.  The 
action  of  the  suprarenal  gland  extract  in  causing  secretion 
is  believed  to  be  upon  the  terminations  of  the  sympathetic 
fibres  in  the  gland  cells.  (It  is  not  always  possible  to  get 
a  secretion  of  saliva  by  the  injection  of  suprarenal  extract.) 

12.  After  the  effects  of  the  suprarenal  extract  have 
passed  oft",  start  the  infusion  of  the  secretin,  giving  it  quite 
slowly.  In  a  minute  or  two  the  pancreas  should  begin  to 
secrete  actively.  When  the  secretion  is  well  established  and 
while  the  secretin  is  still  entering  the  vein  inject  into  the 
opposite  femoral  vein  suprarenal  extract  as  was  done  in  ii. 
What  effect  has  this  upon  the  pancreatic  secretion  and  what 


264  LABORATORY  GUIDE  IN 

explanation  can  you  suggest  for  it,  knowing  the  effect  of 
the  drug  upon  the  blood  vessels? 

13.  Tr}  the  effect  of  an  injection  of  10  mg.  of  nicotine. 
The  influence  of  this  injection  may  not  be  very  marked  on 
account  of  the  earlier  use  of  nicotine  in  the  experiment. 

14.  Give  an  injection  of  pituitary  extract  (0.5-1  cc. 
pituitrin).  These  last  two  drugs  also  cause  marked  con- 
striction of  the  abdominal  vessels. 

The  action  of  the  hormone,  secretin,  upon  the  pancreas  is 
in  marked  contrast  to  that  of  pilocarpine  which  is  so  active 
in  producing  a  secretion  from  the  salivaiy  gland,  and  it 
illustrates  probably  the  relative  importance  of  chemical  and 
nervous  control  over  the  secretion  of  this  gland.  Any  drug 
which  constricts  the  vessels  of  the  gland  and  thus  cuts  off 
its  blood  supply  will  in  this  way  inhibit  its  activity. 


BXPBRIMBNTAL  PHARMACOLOGY  265 


266  LABORATORY  GUIDE  IN 


.  EXPERIMENTAL  PHARMACOLOGY  267 

Antipyretics. 

The  temperature  of  the  animal  body  is  determined  by  the 
relation  between  heat  formation  and  heat  dissipation.  To 
maintain  a  uniform  temperature  in  warm  blood  animals,  it 
is  necessary,  therefore,  to  have  some  mechanism  to  preserve 
a  balance  between  these  two  activities.  This  heat  regulating 
mechanism  has  not  been  shown  to  be  an  anatomical  or 
physiological  unit  or  definite  center,  but  on  the  other  hand 
some  of  the  well-known  centers  are  closely  connected  with 
heat  control. 

Temperature  changes  in  the  warm  blooded  animal  are 
usually  brought  about  by  a  disturbance  of  the  regulating 
mechanism.  For  instance,  the  temperature  may  be  lowered 
by  depressing  the  central  nervous  system  as  by  ether,  chloro- 
form or  chloral,  under  the  influence  of  which  less  heat  is 
formed;  or  the  temperature  may  be  lowered  by  increasing 
heat  output  either  by  dilating  the  peripheral  vessels  and  ex- 
posing more  blood  to  the  outside  air  or  by  increasing  the 
production  of  sweat.  The  typical  antipyretics,  such  as 
acetanihde  and  acetphenetidine,  act  after  this  manner  in- 
creasing output.  They  have  practically  no  effect  against 
normal  temperature,  acting  only  in  fever.  Another  drug 
which  lowers  temperature  is  quinine  and  this  drug  acts  di- 
rectly upon  metabolism  lessening  the  formation  of  heat. 

Drugs  sucli  as  strychnine  or  atropine  which  increase 
muscular  movements,  may  raise  the  temperature,  but  the 
most  important  agents  to  increase  temperature  are  the 
bacterial  poisons  and  toxins. 

The  action  of  the  antipyretics  may  be  studied  in  the  fol- 
lowing experiments : — 


268  LABORATORY  GUIDE  IN 

Select  three  rabbits  of  about  the  same  size,  weighing  say 
1500  G.  to  2000  G.,  and  take  the  rectal  temperature  of  each. 
The  thermometer  bulb  should  be  vaselined  in  each  case  be- 
fore use  and  should  be  inserted  always  about  the  same  dis- 
tance— say  2  inches.  As  the  fever  takes  some  time  to  de- 
velop, for  afternoon  use,  two  of  the  three  rabbits  should  be 
injected  subcutaneously  at  9  A.  M.  with  Witte's  Peptone 
I  G.  per  kilogram  of  body  weight.  The  temperature  should 
be  taken  each  hour  and  plotted  upon  a  chart.  By  afternoon 
both  animals  should  have  a  well  developed  fever.  Inject 
subcutaneously  into  one  of  these  animals  200  mg.  antipyrine 
and  use  the  other  fe/ered  animal  as  a  control.  Into  the 
third  rabbit,  vvhich  is  normal,  inject  200  mg.  antipyrine  and 
this  animal  will  then  serve  as  a  further  control  in  the  ex- 
periment for  the  effect  of  the  drug.  The  temperatures  of 
the  three  animals  should  then  be  taken  every  half  hour 
during  the  afternoon. 

In  place  of  using  peptone  to  produce  the  fever,  it  is  often 
possible  to  secure  even  more  satisfactory  results  by  the  use 
of  the  increase  in  temperature  which  occurs  in  the  ana- 
phylactic reaction.  To  utilize  this  phenomenon,  two  rabbits 
should  be  sensitized  by  the  injection  of  2  cc.  of  beef  serum 
two  weeks  before  they  are  needed  for  the  experiment  in 
fever.  On  the  day  of  the  experiment  at  about  12  o'clock 
(for  afternoon  work)  each  of  the  two  rabbits  should  be  in- 
jected in  the  ear  vein  with  i  cc.  of  a  i-io  solution  of  beef 
serum.  By  i  :30  each  of  the  rabbits  will  have  fever.  One 
of  these  fevered  animals  and  a  third  (normal)  rabbit  are 
now  injected  with  200  mg.  antipyrine  and  the  temperature 
of  each  animal  is  taken  every  half  hour  and  plotted  on 
a  chart  as  described  in  the  earlier  experiment.     One  of  the 


BXPBRIMHNTAL  pharmacology  269 


270  LABORATORY  GUIDB  IN 


EXPERIMENTAL  PHARMACOLOGY 


271 


animals  will  show  the  normal  fever  curve;  the  second  will 
show  the  curve  as  modified  by  the  antipyretic,  and  the  third 
curve  will  show  the  absence  of  effect  in  the  case  of  the  use 
of  an  antipyretic  in  an  animal  with  a  normal  temperature. 

Taste  acetanilide,  acetphenetidine  and  antipyrine,  and  test 
the  solubility  of  each  in  water.  How  would  they  be  pre- 
scribed ? 


272  LABORATORY  GUIDE  IN 

Biological  Assay. 

One  of  the  essential  requirements  in  any  medicine  which 
is  to  be  used  in  the  treatment  of  disease  is  that  not  only  shall 
it  possess  the  properties  usually  found  in  that  drug,  but  also 
that  it  shall  be  of  a  definite  and  uniform  strength.  With 
a  great  many  drugs,  such  as  opium  and  nux  vomica  the 
active  principicc  are  alkaloids  and  are  well  known,  and  in 
such  cases  unifoim  preparations  are  obtainable  by  chemical 
means  as  these  alkaloids  can  be  isolated  in  pure  form  and 
weighed.  For  instance,  the  Pharmacopoeia  directs  that  lOO 
cc.  of  Tincture  of  Opium  shall  contain  i  Gram  of  anhydrous 
morphine  and  lOO  cc.  of  tincture  of  nux  vomica  shall  con- 
tain 0.25  grams  of  the  alkaloids  of  the  drug,  etc.  With  other 
drugs,  however,  the  active  principles  are  either  not  known, 
or  if  known,  it  frequently  happens  that  they  cannot  be 
isolated  without  great  difficulty  and  in  the  process  of  isola- 
tion some  of  the  active  substance  may  be  destroyed.  To  this 
group  of  drugs  to  which  chemical  standardization  cannot 
well  be  applied  belong  the  members  of  the  digitalis  group, 
digitalis,  stropnanthus,  squills;  ergot;  cannabis  indica; 
aconite  and  extracts  of  the  suprarenal  and  pituitary  glands. 

For  such  drugs  in  place  of  chemical  standardization  meth- 
ods have  been  introduced  by  which  the  strength  of  prepara- 
tions is  ascertained  by  the  effects  which  they  produce  upon 
animal  tissues.  This  method  is  known  as  Physiological  or 
Biological  Assay  or  Standardization. 

On  account  of  the  importance  of  the  subject,  a  brief  dis- 
cussion may  be  given  of  the  principal  methods  in  use.  Ful- 
ler details  and  directions  for  the  carrying  out  of  the  tests 
may  be  found  in  the  Pharmacopoeia. 


BXPBRIMBNTAL  PHARMACOLOGY  273 


274  LABORATORY  GUIDE  IN 


,    EXPERIMENTAL  PHARMACOLOGY  275 

For  the  standardization  of  digitalis  and  its  allies  frogs 
are  used  more  frequently  than  any  other  animals.  One  of 
the  most  commori  methods  and  the  one  recommended  by 
the  Pharmacopoeia  is  to  ascertain  the  minimum  dose  of 
digitalis  which  will  bring  the  heart  of  a  frog  to  a  systolic 
standstill  in  one  hour.  This  method  has  been  carried  out 
in  the  experiment  under  the  general  subject  of  digitalis 
(Exp.  VI,  page  171).  Fuller  details  are  to  be  found  in 
the  Pharmacopoeia. 

Another  important  method  of  assaying  digitalis  is  to  as- 
certain the  minimal  fatal  dose  for  a  frog  of  a  certain  size, 
and  from  the  knowledge  of  the  strength  of  the  preparation 
thus  gained  adjustments  to  a  standard  strength  are  made. 
This  is  the  so-called  "Toxic"  method. 

In  the  Focke  method  the  time  of  cessation  of  the  circula- 
tion is  observed  when  a  certain  dose  of  digitalis  is  given. 
Again  the  toxic  dose  for  guinea  pigs  and  also  for  cats  are 
obtained  in  still  other  methods  of  assay  and  adjustments 
made  accordingly.  While  the  two  frog  methods  which  were 
mentioned  first  are  probably  the  most  largely  used  of  any, 
yet  each  method  enumerated  has  certain  adherents  who  pre- 
fer it  to  any  other. 

Brgot. 

Ergot  preparations  are  also  standardized  according  to 
biological  metliods  because  on  account  of  the  complexity  of 
its  chemical  constitution  a  standardization  by  chemical 
means  is  impossible.  There  are  two  principal  methods  in 
vogue.  The  first  is  to  test  the  strength  of  a  preparation 
upon  an  isolated  uterus  of  a  guinea  pig  or  other  animal 
comparii:g  its  activity  with  that  of  some  known  standard. 


276  LABORATORY  GUIDE  IN 

The  method  is  described  in  the  chapter  on  "Perfusion  of 
Isolated  Organs"  (Page  232). 

The  second  method  for  ergot  depends  upon  the  darkening 
or  cyanosis  which  is  produced  in  the  cock's  comb  by  ergot 
preparations. 

Experiment.  Select  preferably  Leghorn  roosters  weighing 
about  1000  G.  to  1200  G.  and  inject  into  the  breast  muscle  of 
one  a  dose  of  the  standard  preparation  which  is  known  to 
produce  a  mild  degree  of  cyanosis  of  the  comb.  Into  the 
breast  muscle  of  the  second  rooster  inject  a  corresponding 
dose  of  the  unknown  preparation  and  compare  the  intensity 
of  the  discoloration  in  the  combs  of  the  two  birds  at  the  end 
of  one  hour.  This  should  give  some  idea  as  to  ihe  relative 
activity  of  the  two  preparations.  Two  or  three  days  later 
inject  the  birds  again  reversing  the  order  of  the  preparations 
upon  the  two  birds  and  giving  doses  modified  by  the  experi- 
ence gained  by  the  first  injection  so  that  they  shall  give  re- 
sults which  are  more  comparable.  In  subsequent  injections 
the  doses  are  narrowed  down  still  further  until  it  is  known 
exactly  how  much  of  the  solution  of  unknown  strength  it  is 
necessary  to  give  to  produce  results  analogous  to  those  ob- 
tained from  a  certain  dose  of  the  standard.  Knowing  this 
relation  it  is  easy  to  calculate  how  much  the  unknown  solu- 
tion must  be  diluted  or  concentrated  to  conform  to  the  stand- 
ard adopted.  The  results  obtained  by  the  isolated  uterus 
method  and  by  the  cock's  comb  method  run  roughly  parallel 
to  each  other. 

Aconite. 

This  drug  is  frequently  assayed  biologically  as  its  activity 
depends  upon  the  presence  of  alkaloids  differing  from  each 
other  in  strength.     In  order,  therefore,  to  get  an  estimate 


BXPBRIMBNTAL  PHARMACOLOGY  277 


278  LABORATORY  GUIDB  IN 


BXPBRIMBNTAL  PHARMACOLOGY  279 

of  the  activity  of  the  total  alkaloids  biological  methods  are 
necessary.  The  method  employed  is  to  ascertain  the  min- 
imal fatal  dose  of  the  aconite  preparation  for  a  guinea  pig. 
The  standard  lethal  dose  adopted  by  the  Pharmacopoeia  for 
the  tincture  of  aconite  is  0.0004  cc  per  gram  of  body  weight 
of  guinea  pig.  The  details  of  the  assay  as  described  in  the 
Pharmacopoeia  were  carried  out  in  the  experimental  work 
on  Aconite  (Page  183). 

Cannabis  Indica. 

The  assay  of  cannabis  depends  upon  the  fact  that  this  drug 
produces  in  dogs  certain  symptoms  of  muscular  inco -ordina- 
tion. The  method  therefore  consists  of  ascertaining  the 
dose  of  a  preparation  which  will  produce  these  symptoms 
and  then  adjusting  its  strength  to  the  standard  requirements. 
The  directions  for  carrying  out  the  test  are  to  be  sought  in 
the  Pharmacopoeia  and  a  preparation  of  cannabis  is  to  be 
assayed  according  to  those  directions. 

Suprarenal  Gland. 

The  assay  of  products  of  the  suprarenal  gland  is  carried 
out  by  means  of  a  comparison  of  the  blood  pressure  rise 
produced  in  dogs  by  an  extract  of  the  glands  with  that  pro- 
duced by  a  solution  of  known  strength  of  the  active  princi- 
ple of  the  gland.  According  to  the  standard  adopted  each 
gram  of  the  dried  suprarenals  shall  contain  the  equivalent 
of  10  mg.  of  the  active  principle  of  the  gland. 

The  directions  for  carrying  out  this  test  are  given  in  the 
Pharmacopoeia. 


28o  LABORATORY  GUIDE  IN 

Pituitary  Gland. 

The  active  principle  of  this  gland  has  not  been  isolated  in 
pure  form  and  therefore  products  made  from  the  gland  have 
to  be  assayed  biologically.  The  method  which  is  used  almost 
exclusively  is  the  isolated  uterus  method  which  as  men- 
tioned above  is  used  for  ergot  assay  and  which  is  described 
in  the  chapter  on  "Perfusion  of  isolated  organs"  (page  232). 

The  Pharmacopoeia  directs  that  1  cc.  of  the  solution  of 
the  Pituitary  Gland  shall  have  the  same  activity  on  the  iso- 
lated uterus  of  a  virgin  guinea  pig  as  a  i  to  20  million  solu- 
tion of  beta-iminazolyl-ethylamine  hydrochloride. 

A  second  method  which  is  not  so  commonly  employed  is 
similar  to  that  in  use  for  suprarenal  glandular  products.  A 
comparison  is  made  of  the  rise  in  blood  pressure  produced 
by  a  certain  dose  of  the  product  to  be  a.-^sayed  with  that 
produced  by  a  standard  preparation. 


nXPURIMBNTAL  PHARMACOLOGY  281 


82  LABORATORY  GUIDE  IN 


2»2 


EXPERIMENTAL  PHARMACOLOGY  283 

Pilocarpus. 

Pilocarpine  is  an  alkaloid  derived  from  the  leaves  of  sev- 
eral species  of  Pilocarpus.  It  has  been  employed  in  certain 
of  the  earlier  experiments,  which  may  be  summarized  in 
order  to  give  a  more  complete  picture  of  the  action  of  the 
drug. 

Describe  the  effects  of  pilocarpine  on — 

the  frog's  heart  (Exp.  page  187)  ; 
the  turtle's  heart  (Exp.  page  191)  ; 
the  pupil  of  the  eye  (Exp.  page  247)  ; 
the  salivary  secretion  (Exp.  page  255)  ; 
and  on  the  intestinal  peristalsis  (Exp.  page  247). 
How  does  atropine  affect  the  activity  of  the  drug? 
I.      (These  drugs  will  be  furnished  you.)     Take  c.003  G. 
of   pilocarpine   hydrochloride  and   describe   any   symptoms 
noted.    Should  any  of  these  become  unpleasant  take  atropine 
sulphate  0.0005  G.  and  describe  any  changes. 

Other  drugs  which  resemble  pilocarpine  in  many  of  their 
actions  are  Muscarine,  from  one  of  the  poisonous  mush- 
rooms (Agaricus  muscarius),  and  Physostigmine  or  Hs- 
serine,  from  the  Calabar  or  Ordeal  bean  (Physostigma 
venenosum) . 

Examine  the  leaves  of  Pilocarpus  jaborandi,  and  the 
calabar  bean. 

Therapeutic  Uses. 
The  employment  of  pilocarpine  in  diseases  of  the  eye  is 
discussed  elsewhere   (page  251)."    The  drug  is  practically 
never  used  for  its  cardiac  action. 

It  is  used  to  increase  the  activity  of  the  sweat  glands 
(diaphoretic  action)   in  various  diseases  in  which  there  is 


284  LABORATORY  GUIDB  IN 

an  abnormal  collection  of  fluid  in  the  body.  (Compare  Di- 
uretics, page  244).  Through  its  action  a  very  large  amount 
of  fluid  may  be  excreted  so  that  in  some  cases  the  body  may 
lose  from  four  to  nine  pounds  in  weight  from  a  single  dose 
of  the  drug. 

Pilocarpine  is  employed  in  some  diseases  of  the  kidneys  in 
which  the  urine  excretion  is  lessened,  with  the  idea  of  se- 
curing the  elimination  of  toxic  materials  by  the  sweat  glands. 


EXPERIMENTAL  PHARMACOLOGY  285 


286  LABORATORY  GVIDB  IN 


EXPBRIMBNTAL  PHARMACOLOGY  287 

The  Nitrite  Series. 

This  series  comprises  several  drugs  wliose  action  in  the 
body  is  confined  almost  exclusively  to  the  blood  vessels.  The 
principal  members  are  Auiyl  Nitrite,  Nitroglycerin,  Sodium 
and  Potassium  Nitrite. 

They  have  been  employed  in  various  experiments  and  the 
results  are  to  be  collected  and  summarized. 

Describe  the  effects  of  the  nitrites  on — 

the  mammalian  blood  pressure  (Exp.  page  207)  ; 
perfused  vessels  (Exp.  page  227)  ; 
on  the  vessels  of  the  intestine  (Exp.  page  299)  ; 
and  on  the  kidney  vessels  (Exp.  page  304). 

Inhale  3  drops  of  amyl  nitrite. 

Therapeutic  Uses. 

The  members  of  this  series  are  employed  in  medicine  to 
lower  the  blood  pressure  and  to  relieve  vascular  spasm. 


288  LABORATORY  GUIDE  IN 

Suprarenal  Gland  Extract. 

The  suprarenal  glands  contain  a  very  active  substance. 
Epinephrine,  which  can  be  isolated,  but  only  with  consider- 
able difficulty. 

For  experimental  work  the  commercial  adrenalin  chloride  (i-iooo)  may  be 
used.  From  one  to  three  drops  of  this  preparation  diluted  with  salt  solution, 
give    good    results. 

Describe  the  effects  of  extract  of  suprarenal  gland  upon 

the  blood  pressure  (Exp,  page  200)  ; 

the  mammalian  heart  (Exp.  page  207)  ; 

perfused  vessels  (Exp.  page  227)  ; 

the  salivary  secretion  (Exp.  page  255)  ; 

the  kidney  vessels  (Exp.  page  304)  ; 

and  upon  intestinal  vessels  (Exp.  page  299). 

I.  Examine  the  local  effects  of  the  drug  by  putting  one 
drop  of  adrenalin  chloride  (i-iooo)  in  the  eye.  Compare 
with  the  normal  eye.  How  would  this  experiment  aid  you 
in  explaining  the  results  obtained  in  the  experiment  on  blood 
pressure? 

Therapeutic  Uses. 

The  powerful  effects  of  the  drug  on  the  circulation  are 
limited  in  their  application  to  therapeutics  by  their  transitory 
nature  and  by  the  fact  that  in  order  to  act  on  the  heart  or 
blood  vessels  (except  the  local  action),  the  drug  has  to  be 
injected  intravenously.  In  some  cases  of  shock  and  collapse 
it  has  been  given  in  this  way,  but  it  requires  constant  admin- 
istration as  the  effects  pass  off  so  rapidly.  It  is  of  most 
value  for  local  application  in  surgical  operations  to  constrict 
the  vessels  and  thus  render  the  field  of  operation  bloodless. 


MXPBRIMBNTAL  PHARMACOLOGY 


2go  LABORATORY  GUIDE  IN 


nXPBRIMENTAL  PHARMACOLOGY  291 

Pituitary  Extracts. 

Extracts  of  the  posterior  lobe  of  the  pituitary  gland  pos- 
sess quite  a  powerful  action  which  is  exerted  mainly  upon 
structures  containing  smooth  muscle.  Their  most  important 
cfiect  perhaps  is  upon  the  blood  vessels  where  a  stimulant 
action  leads  to  an  increased  blood  pressure.  They  also 
cause  contractions  in  other  organs  especially  in  the  uterus 
and  bladder.  The  action  is  probably  not  upon  any  nerve 
structure  but  is  direct  upon  the  muscle  cell. 

The  effect  of  extracts  upon  the  circulation  is  studied  in 
the  experiment  on  page  207. 

The  action  on  smooth  muscle  is  studied  in  the  experiment 
on  page  232. 

Inasmuch  as  extracts  of  the  gland  differ  considerably  in 
strength,  it  is  very  desirable  that  some  method  of  assay  and 
of  standardization  should  be  utilized  in  their  manufacture  in 
order  to  insure  a  uniform  strength  in  the  glandular  products. 
It  is  impossible  to  standardize  them  by  chemical  means  at 
present  on  account  of  our  lack  of  knowledge  of  the  chem- 
istry of  the  active  principle  of  the  gland.  However,  biol- 
ogical methods  of  assay  have  been  adopted  to  a  greater  or 
less  extent,  and  these  are  described  in  the  special  chapter 
devoted  to  the  subject.    Page  280. 


292  LABORATORY  GUIDE  IN 

The  following  experiments  are  not  suitable  for  the  regu- 
lar laboratory  course,  but  may  be  carried  out  by  advanced 
students ;  they  will  also  serve  very  well  for  class  demonstra- 
tions by  the  instructor  in  charge. 

Intestinal  Peristalsis.^ 

The  peristaltic  movements  of  the  intestines  and  the  effects 
of  various  drugs  upon  them  may  be  demonstrated  very  well 
by  submerging  an  animal  in  a  saline  solution,  opening  the 
abdomen  and  keeping  the  intestines  under  the  surface  of  the 
water  by  means  of  a  pane  of  glass. 

For  this  experiment  a  metallic  tank  is  needed  which  will 
be  large  enough  to  allow  a  rabbit  to  be  placed  in  it  when  the 
animal  is  tied  to  an  operating  board.  A  tank  measuring 
70x30x30  cm.  is  large  enough  for  most  purposes  and  it 
should  be  mounted  on  legs  long  enough  to  allow  a  Bunsen 
burner  to  be  placed  under  it.  The  operating  board  should 
be  weighted  with  lead  so  that  it  will  sink  in  the  water.  A 
pane  of  glass  to  cover  the  intestines  and  some  small  lead 
piping,  which  can  be  bent  over  the  sides  of  the  tank  to  act 
as  a  support  for  the  glass,  and  a  bath  thermometer  complete 
the  special  apparatus  necessary  for  the  experiment. 

Fill  the  tank  about  two-thirds  full  of  water  and  add 
sodium  chloride  to  make  a  physiological  salt  solution  (8  G. 
per  liter) .  Light  the  Bunsen  burner  under  the  tank  and  heat 
the  solution  until  it  is  of  the  body  temperature. 


^  This  experiment  may  have  been  carried  out  in  connection  with  the  experi- 
ment on  the  cervical  sympathetic  nerve  in  which  case  it  would  not  be  neces- 
sary to  repeat  it.  The  directions  herein  given,  however,  serve  very  well  in 
case  the  experiment  has  not  been  carried  out  earlier   in  the  course. 


MXPBRIMBNTAL  PHARMACOLOGY  293 


294  LABORATORY  GUIDB  IN 


■EXPERIMENTAL  PHARMACOLOGY  295 

Operation. 

In  the  meantime  ansethetize  a  rabbit  or  cat  in  the  usual 
manner  and  when  anaesthesia  is  complete  insert  a  venous 
cannula  in  the  jugular  vein  and  also  insert  a  tracheal  can- 
nula. Place  short  pieces  of  rubber  tubing  on  both  free  ends 
of  the  tracheal  cannula  and  close  one  by  means  of  a  clamp. 
The  second  piece  of  tubing  is  to  extend  above  the  surface  of 
the  water  when  the  animal  is  submerged. 

Place  the  animal  in  the  Avarm  salt  solution  which  should 
completely  cover  its  body.  Take  great  care  no  water  enters 
the  tracheal  tube,  the  open  end  of  which  is  to  be  tied  to  a 
support  above  the  tank.  Make  an  incision  through  the  skin 
and  muscles  in  the  median  line  of  the  abdomen  from  the  end 
of  the  sternum  to  the  symphysis.  The  abdominal  walls  may 
be  drawn  outward  and  secured  to  the  operating  board  by 
means  of  tacks.  The  intestines  will  float  to  the  top  of  the 
water  and  they  must  be  held  just  beneath  the  surface  by 
placing  the  pane  of  glass  over  them  supporting  the  latter  by 
means  of  the  lead  tubing. 

The  peristaltic  movements  may  now  be  observed  as  w^ell 
as  the  vascular  changes  which  will  follow  the  injection  of 
the  drugs  named  below ;  the  changes  in  color  of  the  intestines 
indicate  the  alterations  in  the  circulation  of  tlie  biood. 

Inject  into  the  jugular  vein  the  following  drugs  in  the 
order  named : — • 

Nicotine  chloride,  5  mg., 
Pilocarpine  hydrochloride,  5  mg., 
Atropine  sulphate,  i  mg., 

Physostigmine    hydrochloride,    3    mg.,    injected 
slowly.     (Observe  any  changes  in  the  skeletal 


296  LABORATORY  GUIDE  IN 

muscles  such  as  twitching.  This  injection  may- 
have  to  be  repeated  to  secure  well  marked  re- 
sults.) 

Adrenalin  chloride  (i-iooo  sol.),  2  drops  in 
salt  solution. 

Amyl  nitrite,  2  or  3  drops  given  by  inhalation. 


MXPBRIMBNTAL  PHARMACOLOGY  297 


LABORATORY  GUIDE  IN 


.EXPERIMENTAL  PHARMACOLOGY  299 

Vasomotor  Changes  in  the  Intestine. 

The  special  apparatus  needed  for  this  experiment  is,  first, 
some  form  of  intestinal  plethysmograph  and  second,  an  in- 
strument to  record  the  intestinal  vascular  changes  upon  a 
blackened  drum. 

The  plethysmograph  may  be  made  very  easily  of  plaster 
of  Paris  after  the  pattern  devised  by  A.  Edmunds.^ 

With  the  plaster  make  a  hemispherical  basin  ten  centi- 
meters in  diameter  and  four  deep  with  the  walls  about  seven 
millimeters  thick  and  with  an  elliptical  opening  in  the  bottom 
of  the  basin  three  centimeters  by  one  and  a  half.  Make  a 
small  opening  in  one  side  of  the  apparatus  and  cement  in  it 
a  short  piece  of  glass  tubing  which  connects  with  the  interior 
of  the  basin  and  which  extends  outside  three  or  four  centi- 
meters to  allow  of  rubber  pressure  tubing  being  connected 
with  it.  Smooth  off  the  upper  edge  of  the  apparatus  with 
sand  paper  so  that  a  piece  of  glass  will  fit  fairly  tightly  on  it. 
A  plethysmograph  made  in  this  way  will  answer  all  the  pur- 
poses of  the  experiment.  Its  more  complicated  forms  may 
be  made  as  described  in  the  article  referred  to.  A  glass  plate 
which  will  close  the  top  of  the  plethysmograph  is  needed  as 
well  as  a  piece  of  rubber  tubing  long  enough  to  connect  the 
plethysmograph  with  the  recording  apparatus,  which  may  be 
either  a  tambour,  a  delicate  piston  recorder  or  a  Brodie  bel- 
lows recorder.  Either  of  these  should  be  provided  with  a 
side  tube  to  allow  of  proper  equalization  of  pressure  on  both 
sides  of  the  piston  or  membrance  of  the  recorder. 


'Jour,    of   Physiol.,   Vol.    XXII,    189S,   page   380. 


300  LABORATORY  GUIDE  IN 

Vaseline  is  needed  as  well  as  a  petrolate  which  melts  at 
a  higher  temperature  than  vaseline  and  which  may  be  made 
by  melting  the  vaseline  with  paraffine  using  such  proportions 
that  the  resulting  mixture  is  fairly  hard  when  cold. 

Operation. 

Anaesthetize  a  cat  or  a  rabbit  as  usual,  tie  it  on  an  operat- 
ing board  and  insert  a  venous  cannula. 

If  it  is  desired  to  take  a  tracing  of  the  carotid  blood  pres- 
sure at  the  same  time,  the  artery  must  be  isolated,  a  cannula 
inserted  and  connected  with  the  mercury  manometer  accord- 
ing to  the  directions  given  on  page  200.  By  thus  taking 
simultaneous  tracings  of  the  general  blood  pressure  and  of 
the  vascular  conditions  in  the  intestinal  area  very  instructive 
records  may  be  obtained. 

It  is  best  to  warm  the  plethysmograph  slightly  before  ap- 
plying it  and  to  have  plenty  of  hot  salt  solution  ready  for  use 
as  well  as  some  absorbent  cotton.  Make  an  incision  about 
4  cm.  long  in  the  median  line  of  the  abdomen  about  midway 
between  the  sternum  and  symphysis  and  with  the  fingers 
carefully  pull  out  of  the  abdominal  cavity  a  short  loop  of  the 
small  intestine.  Place  the  elliptical  opening  of  the  plethys- 
mograph over  the  incision  and  intestinal  loop  and  support  the 
instrument  in  place  with  an  iron  ring.  Draw  the  loop  of  the 
intestine  into  the  apparatus  until  a  section  from  20  to  30  cm. 
long  is  lying  in  the  basin.  Cover  the  intestine  temporarily 
with  absorbent  cotton  dipped  in  the  hot  salt  solution.  Ligate 
doubly  each  end  of  the  section  of  intestine  and  cut  across  it 
between  the  ligatures,  tearing  down  the  mesentery  as  far  as 
possible  and  ligating  any  bleeding  vessels.  Now  return  to 
abdominal  cavity  the  upper  and  lower  attached  ends  of  the 


,nXPBRIMENTAL  PHARMACOLOGY  301 


302  LABORATORY  GUIDE  IN 


■EXPERIMENTAL  PHARMACOLOGY  303 

the  intestine,  leaving  the  isolated  loop  in  the  plethysmograph 
and  connected  to  the  animal  by  its  section  of  mesentery  con- 
taining the  vessels  and  nerves. 

Pack  absorbent  cotton  rubbed  up  w^ith  the  hardened 
vaseline  around  the  mesentery  so  as  to  completely  close  the 
elliptical  opening,  using  soft  vaseline  to  make  it  air  tight 
near  the  vessels ;  'great  care  must  be  taken  not  to  constrict 
the  vessels.  Remove  the  cotton  covering  from  the  intestines ; 
vaseline  the  upper  rim  of  the  plethysmograph  and  close  it 
with  the  pane  of  glass  which  has  been  dipped  in  warm  salt 
solution  to  prevent  the  condensation  of  moisture  on  its  sur- 
face. 

Connect  the  instrument  with  the  recorder,  close  the  side 
tube  on  the  latter  and  take  a  tracing,  which,  if  the  experi- 
ment has  been  properly  carried  out,  will  show  both  cardiac 
beats  and  the  respiratory  waves.  Arrange  the  lever  to  write 
in  line  with  the  blood  pressure  pointer  and  the  time  marker 
which  has  been  placed  below. 

Take  tracings  to  shoAV  the  effects  of  the  following  drugs 
on  the  general  arterial  blood  pressure  and  on  the  vessels  of 
the  splanchnic  area. 

Adrenalin  chloride  (i-iooo),  1-3  drops  in  salt  solution. 

Digitalis,  tincture,  i  cc.  in  salt  solution. 

Nicotine  chloride,  2  mg.  in  salt  solution. 

Amyl  nitrite,  2  drops  tc  be  given  by  inhalation. 

Pituitary  extract  (Pituitrin),  0.5  cc.  in  salt  solution. 

Chloroform  to  be  given  until  the  heart  stops. 

The  tracing  from  this  experiment  should  be  studied  in 
connection  with  those  obtained  earlier  in  the  course  in  the 
experiment  upon  the  dog's  heart  and  with  the  results  found 
Vvhen  the  vessels  of  the  frog  were  perfused  Vv^ith  salt  solu- 
tion. 


304  LABORATORY  GUIDE  IN 

Vascular  Changes  in  the  Kidney. 

To  record  the  changes  taking  place  in  the  size  of  the  kid- 
ney due  to  the  differences  in  its  blood  content,  an  oncometer 
and  a  recording  apparatus  are  needed.  The  former  can. be 
made  from  plaster  of  Paris^  in  the  same  manner  as  the  intes- 
tinal plethysmograph.  Several  sizes  should  be  made  to  ac- 
commodate the  kidneys  of  dift'erent  sized  animals.  A  cylin- 
drical-shaped oncometer  with  a  flat  base,  having  a  diameter 
of  about  5  cm.  and  with  walls  about  3.5  cm.  high  is  large 
enough  for  the  average  sized  cat  or  rabbit.  One  side  should 
have  a  cut  in  it  about  0.5  cm.  wide  and  extending  nearly  to 
the  bottom  of  the  apparatus,  where  the  cut  should  be  widened 
to  allow  greater  room  for  packing  around  the  pedicle  of  the 
kidney.  On  the  opposite  side  of  the  oncometer  is  a  glass 
tube  communicating  with  the  interior  of  the  apparatus  and 
extending  outward  for  the  attachment  of  rubber  pressure 
tubing.  A  piece  of  glass  is  needed  to  fit  tightly  the  top  of 
the  instrument.  Vaseline  together  with  a  mixture  of  vaseline 
and  wax  (as  described  on  page  300)  is  also  necessary. 

For  recording  the  volume  changes  on  the  kymograph,  a 
tambour,  piston  recorder,  or  a  bellows  recorder  as  mentioned 
under  the  experiment  on  the  intestinal  vessels  (page  299) 
is  required.    It  should  be  fitted  as  before  with  a  side  tube. 


^  In  place  of  the  oncometer  made  of  plaster  of  Paris  in  the  manner  de- 
scribed a  Roy  kidney  oncometer  such  as  can  be  obtained  on  the  market  may  be 
used,  thus  saving  considerable  time  as  it  can  be  more  easily  adjusted  than  the 
plaster  apparatus.  Descriptions  of  the  Roy  oncometer  may  be  found 
in    works    on    physiology. 


-  BXPBRIMBNTAL  PHARMACOLOGY  305 


3o6  LABORATORY  GUIDE  IN 


-    nXPBRIMENTAL  PHARMACOLOGY  307 

Operation. 

Anjesthetize  a  rabbit  or  a  cat  and  insert  one  cannula  in  the 
jugular  vein  and  a  second  in  the  carotid  artery.  Connect  the 
latter  with  the  mercmy  manometer  for  blood  pressure, 
which  has  been  arranged  according  to  the  directions  given 
on  page  200.  Either  kidney  may  be  employed  in  the  experi- 
ment, the  choice  largely  depending  upon  the  arrangement  of 
the  apparatus  and  the  light.  If  the  left  has  been  selected, 
make  an  incision  in  the  abdomen  well  over  in  the  left  flank, 
extending  from  the  costal  margin  downward,  the  length  of 
the  incision  necessary  depending  upon  the  size  of  the  onco- 
meter. Draw  the  side  muscles  outward,  if  necessary  con- 
tinuing the  cut  in  the  muscles  along  the  costal  margin,  and 
nail  the  reflected  muscles  to  the  operating  board.  The  intes- 
tines and  stomach  should  be  packed  back  with  absorbent 
cotton  which  has  been  dipped  in  hot  salt  solution  and  the 
abdominal  cavity  closed  in  as  far  as  possible  with  a  hot 
towel.  This  should  leave  the  left  kidney  well  exposed. 
Loosen  it  very  carefully  from  all  its  fibrous  attachments  and 
remove  the  fat  as  far  as  possible,  so  that  the  kidney  remains 
connected  only  by  the  structures  entering  the  hilus. 

Place  the  organ  in  the  w^armed  oncometer  so  that  the 
pedicle  passes  through  the  widened  end  of  the  cut  in  the 
side  of  the  apparatus.  Pack  around  the  pedicle  with  ab- 
sorbent cotton  saturated  with  the  hardened  vaseline  and 
completely  close  the  opening  in  the  side  in  the  same  way, 
employing  the  soft  vaseline  to  make  it  entirely  air  tight.  Of 
course  it  is  essential  to  see  that  no  undue  pressure  is  exerted 
on  the  kidney  vessels ;  the  circulation  must  remain  perfectly 
free  in  them.  Close  the  top  of  the  oncometer  with  the 
pane  of  glass  which  is  made  air  tight  with  vaseline.     Con- 


3o8  LABORATORY  GUIDE  IN 

nect  the  outlet  tube  of  the  instrument  with  rubber  tubing,, 
the  other  end  of  which  is  connected  with  the  recorder.  Ar- 
range the  writing  point  of  the  recorder  so  that  it  marlcs 
directly  above  the  pointer  of  the  blood  pressure  manometer 
and  the  time  marker.  Close  the  side  tubes  of  the  recorder 
and  the  lever  should  show  the  distinct  cardiac  pulsations  as 
they  are  indicated  by  the  changes  in  the  kidney  volume.  If 
the  pulsations  do  not  show,  either  the  kidney  vessels  are  con- 
stricted or  the  apparatus  is  not  air  tight. 

When  all  the  apparatus  is  working  satisfactorily  inject  the 
following  drugs  into  the  jugular  vein: 

Adrenalin  chloride,  i-iooo,  1-3  drops  in  salt  solution. 

Tincture  of  digitalis,  i  cc.  in  salt  solution. 

Nicotine  chloride,  3  mg.  in  salt  solution. 

Amyl  nitrite,  3  drops,  given  by  inhalation. 

Pituitary  extract  (Pituitrin),  0.5-1  cc.  in  salt  solution. 

Chloroform  to  be  given  until  the  heart  stops. 

These  tracings  are  to  be  studied  after  the  manner  outlined 
in  the  section  on  intestinal  vasomotor  changes  (page  303) 
and  in  connection  with  the  tracing  obtained  in  that  experi- 
ment. 


BXPBRIMBNTAL  PHARMACOLOGY  309 


iio  LABORATORY  GUIDE  IN 


^  BXPBRIMENTAL  PHARMACOLOGY  311 

Perfusion  of  the  Isolated  Frog's  Heart. 

Pith  the  brain  and  the  cord  of  a  large  frog  weighing  about 
75  grams.  Tie  it  on  the  frog  board  and  expose  the  heart 
in  the  usual  manner.  Pass  a  fine  ligature  around  both  aortae. 
With  a  fine  pair  of  scissors  make  a  V-shaped  incision  in  one 
of  the  aortae,  insert  a  cannula  pointing  towards  the  heart 
and  tie  it  in,  tying  both  aortae  at  the  same  time.  With  a 
fine  glass  capillary  pipette  wash  the  blood  out  of  the  cannula 
with  Ringer  solution  until  it  is  clear.  Divide  the  aortae. 
Divide  the  connections  of  the  heart  with  the  posterior  wall 
of  the  thorax  avoiding  cutting  any  of  the  vessels.  Pass  a 
ligature  around  all  the  remaining  vessels  and  turn  the  heart 
up  so  as  to  expose  the  ascending  vena  cava  into  view.  Tie 
a  cannula  into  it  as  low  down  as  possible,  pointing  towards 
the  heart,  and  tying  off  all  the  other  vessels  at  the  same  time. 
Fill  the  cannula  with  Ringer  solution,  and  remove  the  heart 
from  the  body. 

The  perfusion  apparatus  consists  of  a  double  reservoir 
provided  with  Mariotte  stoppers,  rubber  tubing,  and  a  3- 
way  stop-cock.  Place  100  cc.  of  Ringer  solution  in  one 
reservoir,  and  the  same  amount  of  Ringer  solution  to  which 
2  to  3  drops  of  i-iooo  adrenaline  solution  have  been  added, 
in  the  other.  Connect  the  venous  cannula  with  the  per- 
fusion apparatus  making  sure  that  all  air  bubbles  have  been 
excluded.  The  cannula  inserted  into  the  aorta  is  to  be  held 
in  position  by  a  clamp.  The  tip  of  the  ventricle  is  attached 
by  means  of  a  fine  spring  clasp  and  silk  thread  to  a  light 
recording  lever.  The  pressure  of  the  perfusion  fluid  should 
not  exceed  6  cm.,  and  the  flow  of  the  perfusion  fluid  into  the 


312  LABORATORY  GUIDE  IN 

heart  is  adjusted  by  means  of  a  screw  clamp,  and  is  to  be 
maintained  constant  throughout  the  experiment.  A  rate  of 
flow  so  that  1-2  drops  are  expelled  with  each  systole  will 
probably  be  most  satisfactory.  The  resistance  against  which 
the  heart  is  to  contract  is  adjusted  by  properly  tipping  the 
cutflow  cannula,  and  this  should  likewise  be  kept  constant. 

After  obtaining  a  normal  record  of  the  heart  action  turn 
on  the  adrenalin  perfusion  fluid  and  note  the  effects  of  this 
drug  upon  the  heart.  When  this  has  been  noted  turn  on 
Ringer  solution  until  the  heart  has  returned  to  its  normal 
condition.  Now  add  to  the  fresh  Ringer  solution  0.5  cc. 
of  a  i-iooo  solution  of  aconitine  hydrochloride.  Continue 
the  perfusion  with  this  drug  until  the  heart  stops. 

Other  drugs  that  may  be  tried  are : 

Cocaine  hydrochloride,  1-25000. 

Calcium  chloride,  1-500. 

Digitalein,  i -20000. 

Ammonium  chloride,  i-iooo. 


nXPBRIMBNTAL  PHARMACOLOGY  313 


ii4  LABORATORY  GUIDE  IN 


nXPBRlMENTAL  PHARMACOLOGY  315 

The  Action  of  Drugs  on  the  Isolated  Mammalian  Heart. 

The  apparatus  required  for  this  experiment  consists  of  a 
double  reservoir  suspended  at  a  height  of  about  60  inches, 
and  connected  by  means  of  rubber  tubing  and  a  3-way  stop- 
cock with  a  glass  tube  drawn  out  into  a  cannula  and  passing 
through  a  warming  chamber.  The  latter  may  conveniently 
be  made  of  a  small  Liebig  condenser,  the  top  and  bottom  of 
which  have  been  cut  off.  The  bottom  is  closed  with  a  rub- 
ber stopper  through  which  the  inner  cannula  tube  is  passed. 
Through  the  lower  exit  tube  of  the  modified  condenser  a 
brass  rod  is  fitted  (after  the  manner  of  Dale).  The  water 
in  the  warming  jacket  is  maintained  at  the  desired  tem- 
perature by  applying  the  Bunsen  flame  to  the  brass  rod,  and 
this  heats  the  perfusion  fluid  as  it  runs  through  the  inner 
tube. 

Operation. 

Anaesthetize  a  small  rabbit  or  a  cat  with  ether.  Expose 
the  carotid  artery  and  insert  into  it  a  cannula.  Bleed  the  ani- 
mal into  a  large  evaporating  dish  and  defibrinate  the  blood 
thoroughly  by  whipping  with  glass  rods.  Quickly  open  the 
animal's  chest,  open  the  pericardium,  and  while  the  heart  is 
still  beating  seize  the  aorta'  with  a  fine  pair  of  forceps  and 
divide  it  at  the  point  where  its  branches  begin  to  come  oft'. 
Sever  all  the  other  vessels,  and  at  once  transfer  it  to  Ringer- 
Locke  solution.  It  will  usually  beat  quite  vigorously  at  this 
time.  If  it  beats  feebly  gentle  massage  with  the  fingers  in 
the  Ringer-Locke  solution  will  help  to  empty  the  heart  and 
its  vessels  of  blood  w^hich  is  essential  to  prevent  clotting  in 
the  coronaries.  The  heart  is  now  left  in  the  solution  after 
the  aorta  and  the  pulmonary  artery  have  been  identified, 


3i6  LABORATORY  GUIDE  IN 

until  such  time  as  you  are  ready  to  proceed  with  the  experi- 
ment. 

The  defibrinated  blood  is  strained  through  cotton  and 
diluted  with  warmed  (38°C.)  Ringer-Locke  solution  to 
make  a  total  volume  of  about  200  cc.  (From  10  to  15% 
blood  will  give  best  results.)  Pass  a  stream  of  oxygen 
through  the  diluted  blood  for  a  few  minutes.  To  100  cc.  of 
the  oxygenated  blood  solution  add  0.2  cc.  of  i-iooo  solu- 
tion of  ouabain  and  transfer  this  to  one  reservoir.  Into  the 
other  reservoir  place  100  cc.  of  normal  oxygenated  blood. 
Fill  the  whole  system  with  the  perfusion  fluids,  excluding 
rigorously  all  air  bubbles.  Tie  the  cannula  of  the  perfusion 
apparatus  into  the  aorta,  making  sure  that  it  does  not  pene- 
trate the  semilunar  valves.  Connect  the  apex  of  the  heart 
with  a  recording  lever  by  means  of  a  hook  and  silk  thread, 
and  start  the  perfusion  of  the  coronaries  with  the  normal 
blood  solution  at  a  constant  temperature  of  38°  C.  The 
perfusion  fluid  is  to  be  caught  in  a  large  beaker  and  is  to  be 
returned  to  the  reservoir  as  often  as  may  be  necessary. 
At  the  beginning  of  the  experiment  the  perfusion  fluid  will 
run  through  rather  rapidly,  and  the  heart  will  probably  beat 
quite  irregularly.  After  the  heart  has  become  perfectly 
regular,  and  a  record  has  been  taken  on  the  revolving  drum, 
2  to  3  drops  of  a  i-ioooo  solution  of  adrenalin  may  be  added 
to  the  normal  perfusion  fluid.  Note  its  effects  upon  the 
heart,  especially  with  regard  to  the  amplitude,  rate  of  beat, 
and  the  rate  of  the  outflow  of  the  perfusion  fluid. 

After  the  effects  of  adrenalin  have  been  studied,  turn  on 
the  ouabain  perfusion  fluid,  and  study  the  various  stages  of 
digitalis  action  upon  the  heart.  Continue  the  perfusion  with 
this  drug  until  the  heart  has  stopped  beating  and  for  5  to  10 
minutes  thereafter. 


EXPERIMENTAL  PHARMACOLOGY  317 


LABORATORY  GUIDB  IN 


EXPERIMENTAL  PHARMACOLOGY  319 


320  LABORATORY  GUIDE  IN 


DRUGS  AND  REAGENTS  REQUIRED  FOR  EACH 
EXPERIMENT. 

Aneesthetics :     Page  ii. 
Ether. 
Chloroform. 
Paraldehyde. 

Chloretone  in   alcohol  or   olive   oil. 
Urethane. 

Morphine    Sulphate. 
Anticoagulating  Solutions:     Page    17. 
Sodium  Sulphate,  half  saturated. 
Sodium  citrate.     5-10%. 
Magnesium   Sulphate,  25%. 
General  Reagents  Needed : 
Alcohol. 
Chloroform. 
Ether. 

Dilute   Sulphuric  Acid. 
Potassium  Carbonate,   10%. 
Potassium  Hydroxide,   10%. 
Alkaloidal  Reagents. 

Tannic  Acid,   10%. 

Picric  Acid,  Saturated. 

Iodine  in  Potassium  Iodide,  U.  S.  P. 

Mercury  Potassium  Iodide,  U.  S.  P. 

Phosphotungstic  Acid,  2%. 
Fehling's  Solution,  U.  S.  P. 
Ferric  Chloride  Solution. 
Concentrated  Acids. 
Alkaloids :     Page  20. 
Quinine. 

Quinine  Sulphate. 
Glucosides :     Page  28. 
Amygdalin. 
Bitter  Almonds. 
Volatile  Oils  :     Page  32. 
Oil  of  Turpentine. 
Cotton  Seed  Oil. 
Resins :     Page  39. 

Powdered  resin. 


LABORATORY  GUIDE  IN 

Saponins :     Page  40. 

Saponin,  2%. 

Castor  Oil. 
Powders :     Page  51. 

Calomel. 

Sugar  of  jMilk. 
Capsules :     Page    55. 

Quinine   Sulphate. 

Oil  of  Turpentine. 
Cachets :     Page  59. 

Quinine   Sulphate. 
Pills :     Page  60. 

Extract  of  Gentian. 

Starch. 

Lycopodium. 
Emulsions :     Page  67. 

Bitter  Almonds. 

Asafoetida. 

Castor  Oil. 

Powdered  Acacia. 
Ointments :     Page    72. 

Extract  of   Belladonna. 

Benzoinated  lard. 

Hydrous  wool  fat. 
Suppositories :     Page    76. 

Oil   of   Theobroma. 

Tannic  Acid. 

Lycopodium. 
Physiology  of   Frog :     Page  80. 

Acid  fuchsin,   1.5%. 
Soporifics :     Page  88. 

Chloral. 

Sulphonal. 

Paraldehyde. 

Veronal. 
Xux   Vomica :     Page   96. 

Powdered  nux  vomica. 

Strychnine   Sulphate. 

Strychnine  Sulphate,  i/io%  Solution. 

Potassium   Bichromate. 

Manganese    Dioxide. 
Camphor  Group;     Page   112. 

Camphor. 

Thujon. 

Picrotoxin. 


EXPERIMENTAL  PHARMACOLOGY  323 

Opium :     Page  116. 

Morphine  Sulphate. 

Cane  Sugar. 

Iodic   Acid,   5%   solution. 

Starch  Paste. 

Codeine   phosphate,    1.5%    solution. 
Curara :     Page  127. 

Curara   Solution. 
Nicotine :     Page    132. 

Nicotine  Chloride  Solution. 

Veratrine :     Page    135. 

Veratrine   Sulphate,  0.1%   solution. 

Caffeine  Group  ;     Page  136. 

Caffeine. 

Sodium  Benzoate. 

Theobromine-Sodium    Salic3'late. 
Cocaine :'    Page   143. 

Cocaine   Hydrochloride. 

Cocaine  Hydrochloride,  4%  solution. 

Belladonna  Group  :     Page   147. 

Powdered    Belladonna    Leaves. 

Atropine  Sulphate. 

Potassium  Hydroxide  in  Alcohol. 

Cinchona:     Page  156. 
Quinine. 

Quinine    Sulphate. 
Quinine    Hydrochloride. 
Chlorine  Water. 

Anesthetics :     Page   160. 
Nitrous   Oxide. 
Ethyl   Chloride. 
Magnesium  Sulphate. 
Sodium  Oxalate. 
Calcium  Chloride,  3%  solution. 

Digitalis   Group :     Page    16S. 

Powdered   Digitalis  leaves. 
Bruised  Digitalis  leaves. 

Aconite :     Page    183. 

Tincture  of  Aconite. 

Drugs   on    Frog's    Heart :     Page    187. 

Pilocarpine    Nitrate,   0.5%    solution. 
Atropine   Sulphate,   0.1%    solution. 


324 


LABORATORY  GUIDE  IN 

Drugs   on   Turtle's   Heart:     Page    191. 

Nicotine  Solution. 

Pilocarpine   Solution. 

Atropine  Solution. 
Drugs  on  Blood  Pressure :     Page  200. 

Amyl  Nitrite. 

Suprarenal  Gland  Extract. 

Nicotine  Chloride  Solution. 

Pituitary   Gland   Extract. 

Tincture  of  Digitalis. 

Barium  Chloride,  2%   solution. 
Drugs  on  Mammalian  Heart :     Page  207. 

Same  as  for  "Blood  Pressure." 
Digitalis  in  Auricular   Fibrillation :     Page   220. 

Tincture  of  Digitalis. 
Perfusion  of  Blood   Vessels :     Page   227. 

Sodium   Nitrite. 

Suprarenal  Gland   Extract. 
Drugs  on  Isolated  Tissues :     Page  232. 

Suprarenal  Gland  Extract. 

Pilocarpine    Solution. 

Atropine  Solution. 

Fluid  Extract  of  Ergot. 

Pituitary  Gland  Extract. 
Diuresis :     Page   239. 

Theocin. 

Sodium    Iodide. 

Sodium  Nitrite,  10%  solution. 

Fehling's   Solution. 

Sodium  Nitrate,  5%  solution. 
Cervical  Sympathetic  System :     Page  247. 

Nicotine  Chloride  Solution. 

Pilocarpine,  0.5%   solution. 

Atropine,  0.1%  solution. 

Suprarenal  Gland  Extract. 
Salivary  and  Pancreatic  Secretion  :     Page  255- 

Same  as  for  Cervical  Sympathetic  System. 

Secretin. 
Antipyretics :     Page   267. 

Peptone. 

Beef  Serum. 

Antipyrine. 

Acetanilide. 

Acetphenetidine. 


EXPERIMENTAL  PHARMACOLOGY.  325 

Biological  Assay  :     Page  272. 

Fluid  Extract  Cannabis. 

Fluid  Extract  Ergot. 

Suprarenal  Gland  Extract. 

Pituitary   Gland   Extract. 
Intestinal  Peristalsis  :     Page  292. 

Same  as  for  Cervical  Sympathetic  System. 

Physostigmine   Hydrochloride. 

Amyl  Nitrite. 
Vasomotor  Changes  in  the  Intestine  :     Page  299. 

Suprarenal  Gland  Extract. 

Tincture  of  Digitalis. 

Nicotine  Chloride   Solution. 

Amyl  Nitrite. 

Pituitary   Gland   Extract. 
Vasomotor  Changes  in  the  Kidney :     Page  304. 

Same  as  for  the  Intestine. 
Isolated  Frog's   Heart:     Page  311. 

Cocaine  Hydrochloride. 

Calcium  Hj'drochloride. 

Digitalein. 

Ammonium   Chloride. 
Isolated   Mammalian   Heart:     Page  315. 

Suprarenal   Extract. 

Ouabain. 


INDEX. 

PAGE 

Absinthe    112 

Ac.id   f uchsin    84 

Aconite 183 

Aconitine    •  •  •  183 

Adrenalin    288 

Alkaloids   ". 20 

Alkaloidal  precipitants 27 

Amygdalin 31 

Amyl   nitrite 287 

Anaesthetics    160 

Ansesthetic  for  cat 12 

for  dog  12 

for    rabbit    11 

Analysis  of  tracings   215 

Antipyretics    267 

Aqua    36 

Assay  of  tincture  of  digitalis   171 

"       "     aconite    184 

Atropine    I47 

Auricular  Fibrillation,   Digitalis   in 220 

Balsams   . 39 

Belladonna  series I47 

Berberine    28 

Biological  assay   272 

Blood  pressure  in  animals,  drugs  on .  .  . .' 200 

Brucine    96 

Cachets   59 

Caffeine    136 

Camphor    112 

Cat  box   12 

Cannabis  assay 279 

Cannulas,  arterial  and  venous 16 

tracheal    18 

Capsules    55 

Chemistry  of  drugs  20 

Chloral  88 

Chloralose 95 

Chloretone   95 

Chloroform    160 


328  INDEX. 

PAGE 

Cinchona   156 

Cocaine    - 143 

Codeine    ; 116 

CofiFee    136 

Coniine 28 

Curara    127 

Curine    127 

Digitalis    168 

Digitalis  in  Auricular  Fibrillation  : 220 

Diuresis    239 

Diuretic,  cardiac 180 

Dover's  powder  124 

Drugs  and  reagents  required  321 

Emulsions    6y 

Epinephrine    288 

Ergot  assay   275 

Eserine    25 1 

Ether    160 

Ethyl  chloride  • 163 

Euquinine    i59 

Fehling's   reaction    28 

Fixed  oil  emulsion 68 

Frog's  heart,  drugs  on 187 

"          "            "        "  isolated    311 

Glucosides    28 

Gums    40 

Gum  resins  40 

Gum  resin  emulsion 68 

Heroine   124 

Homatropine 152 

Hyoscine   I47 

Hyoscyamine   I47 

Hypnotics    88 

Infusion  of  digitalis   176 

Intestine,  Vasomotor  changes  in 299 

Isolated  tissues,  drugs  on 232 


INDEX.  329 

PAGE 

Isolated  frog's  heart,  drugs  on • 311 

Isolated  mammalian  heart,  drugs  on  3i5 

Intestinal  peristalsis  247,  292 

Laudanum    124 

Locke's  solution   I9 

Magnesium  sulphate  anaesthesia   164 

Mammalian  heart,  drugs  on 207 

"     isolated    .' 3i5 

Meconic  acid • 116 

Meconic  acid  reaction    120 

Morphine   , • 116 

■  Muscarine 283 

Mydriatics    252 

Myotics    \ 252 

Nicotine    132 

Nicotine  chloride   132 

Nitrite    series    287 

Nitrous  oxide   163 

Nux  vomica 96 

Oils,  fatty  35 

volatile    32 

Ointments    72 

Oleoresins    39 

Opium    116 

Operations  on  frogs,  pithing 13 

to  inject  in  lymph  sac 13 

to  expose  the  heart 14 

Operations  on  mammals,  to  insert  an  arterial  cannula 17 

to  insert  a  tracheal  cannula  iS 

to  insert  a  venous  cannula   16 

Pancreatic  secretion    255 

Paraldehyde    88 

Paregoric   124 

Percolation  • 44 

Perfusion  of  blood  vessels 227 

Peristalsis,    drugs    on    intestinal    247,  292 

Pharmacopoeia    ^ 9 

Physiological  assay   272 

Physiology  of  frog's  nervous  system 80 


330  INDEX. 

PAGE 

Phj'sostigmine    251 

Picrotoxin    115 

Pills     60 

Pilocarpus    283 

Pipette,  glass  injecting  •. 14 

Pithing   frogs    13 

Pituitary  gland  assay   280 

extracts    291 

Plethysmograph,   intestinal    299 

Powders    51 

Quinine    156 

Reagents  and  drugs  required    321 

Resins    , 39 

Ringer's   solution    19 

Sage    112 

Salivary  secretion,   drugs  on 255 

Saponins    ; 40 

Secretin,  preparation  of 260 

Solutions,  physiological    19 

Anticoagulating    17 

Soporifics   88 

Spiritus   ■ 36 

Squills    168 

Stomach  tube  for  cats   11 

dogs    13 

rabbits    . 1 1 

Strophanthus    168 

Strychnine    96 

Sulphonal    88 

Suppositories    76 

Suprarenal  gland  assay   279 

extract    288 

Sympathetic  system,   drugs   on    247 

Syrupus    2^ 

Tansy    112 

Tetronal    95 

Theobromine 136 

Theocin    240 

Theophyllin     136 


INDEX. 


531 


PAGE 

Thebaine    123 

Thuj  on    112 

Trional    95 

Turtle's  heart,  drugs  on    igi 

Tubocurarine    127 

Tyrode's  solution    _ 19 

Urethane   95 

Vasomotor   effects   in  the  intestine 299 

in  the  kidney  304 

Veratrine    135 

Veronal    ' 88 

Vitali's   test    151 


^^5 


aP'305-  £^s- 


